Recombinant host cell with altered membrane lipid composition

ABSTRACT

The present invention is in the field of recombinant biotechnology, in particular in the field of protein expression. The invention generally relates to a method of expressing a protein of interest (POI) from a host cell. The invention relates particularly to improving a host cell&#39;s capacity to express and/or secrete a protein of interest and use of the host cell for protein expression. The invention also relates to cell culture technology, and more specifically to culturing cells to produce desired molecules for medical purposes or food products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/499,072 filed Sep. 27, 2019, which is a 35 USC § 371 National Stageapplication of International Application No. PCT/EP2018/057853 filedMar. 28, 2018, now expired; which claims the benefit under 35 USC §119(a) to EP Application Serial No. 17163588.1 filed Mar. 29, 2017, nowexpired. The disclosure of each of the prior applications is consideredpart of and is incorporated by reference in the disclosure of thisapplication.

SEQUENCE LISTING

The instant application contains a Sequence Listing which as beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Apr. 25, 2023, isnamed BOE15238PTUSD1_ST26.xml, and is 150,000 bytes in size.

FIELD OF INVENTION

The present invention is in the field of recombinant biotechnology, inparticular in the field of protein expression. The invention generallyrelates to a method of expressing a protein of interest (POI) from ahost cell by over- or underexpressing a polynucleotide encoding aprotein involved in lipid metabolism. The invention relates particularlyto improving a host cell's capacity to express and/or secrete a proteinof interest and use of the host cell for protein expression. Theinvention also relates to cell culture technology, and more specificallyto culturing cells to produce desired molecules for medical purposes orfood products or feed products.

BACKGROUND OF THE INVENTION

Successful production of proteins of interest (POI) has beenaccomplished both with prokaryotic and eukaryotic hosts. The mostprominent examples are bacteria like Escherichia coli, yeasts likeSaccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha,filamentous fungi like Aspergillus awamori or Trichoderma reesei, ormammalian cells like CHO cells. While the yield of some proteins isreadily achieved at high rates, many other proteins are only produced atcomparatively low levels.

Generally, heterologous protein synthesis may be limited at differentlevels. Potential limits are transcription and translation, proteinfolding and, if applicable, secretion, disulfide bridge formation andglycosylation, as well as aggregation and degradation of the targetproteins. Transcription can be enhanced by utilizing strong promoters orincreasing the copy number of the heterologous gene. However, thesemeasures clearly reach a plateau, indication that other bottlenecksdownstream of transcription limit expression.

High level of protein yield in host cells may also be limited at one ormore different steps, like folding, disulfide bond formation,glycosylation, transport within the cell, or release from the cell. Manyof the mechanisms involved are still not fully understood and cannot bepredicted on the basis of the current knowledge of the state-of-the-art,even when the DNA sequence of the entire genome of a host organism isavailable. Moreover, the phenotype of cells producing recombinantproteins in high yields can be decreased growth rate, decreased biomassformation and overall decreased cell fitness.

Various attempts were made in the art for improving production of aprotein of interest, such as overexpressing chaperones which shouldfacilitate protein folding, external supplememtation of amino acids, andthe like.

However, there is still a need for methods to improve a host cell'scapacity to produce and/or secrete proteins of interest. The technicalproblem underlying the present invention is to comply with this need.

The solution of the technical problem is the provision of means, such asengineered host cells, methods and uses applying said means forincreasing the yield of a non-membrane protein of interest in aeukaryotic host cell by over- or underexpressing in said host cell atleast one polynucleotide encoding a protein which is involved in lipidmetabolism. These means, methods and uses are described in detailherein, set out in the claims, exemplified in the Examples andillustrated in the Figures.

Accordingly, the present invention provides new methods and uses toincrease the yield of recombinant proteins in host cells which aresimple and efficient and suitable for use in industrial methods. Thepresent invention also provides host cells to achieve this purpose.

It must be noted that as used herein, the singular forms “a”, “an” and“the” include plural references and vice versa unless the contextclearly indicates otherwise. Thus, for example, a reference to “a hostcell” or “a method” includes one or more of such host cells or methods,respectively, and a reference to “the method” includes equivalent stepsand methods that could be modified or substituted known to those ofordinary skill in the art. Similarly, for example, a reference to“methods” or “host cells” includes “a host cell” or “a method”,respectively.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”. For example, A, B and/or C means A, B, C, A+B, A+C, B+C andA+B+C.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange. It includes also the concrete number, e.g., about 20 includes 20.

The term “less than”, “more than” or “larger than” includes the concretenumber. For example, less than 20 means 20 and more than 20 means ≥20.

Throughout this specification and the claims or items, unless thecontext requires otherwise, the word “comprise” and variations such as“comprises” and “comprising” will be understood to imply the inclusionof a stated integer (or step) or group of integers (or steps). It doesnot exclude any other integer (or step) or group of integers (or steps).When used herein, the term “comprising” can be substituted with“containing”, “composed of”, “including”, “having” or “carrying” andvice versa, by way of example the term “having” can be substituted withthe term “comprising”. When used herein, “consisting of” excludes anyinteger or step not specified in the claim/item. When used herein,“consisting essentially of” does not exclude integers or steps that donot materially affect the basic and novel characteristics of theclaim/item. In each instance herein any of the terms “comprising”,“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms.

Further, in describing representative embodiments of the presentinvention, the specification may have presented the method and/orprocess of the present invention as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.In addition, the claims directed to the method and/or process of thepresent invention should not be limited to the performance of theirsteps in the order written, and one skilled in the art can readilyappreciate that the sequences may be varied and still remain within thespirit and scope of the present invention.

It should be understood that this invention is not limited to theparticular methodology, protocols, material, reagents, and substances,etc., described herein. The terminologies used herein are for thepurpose of describing particular embodiments only and are not intendedto limit the scope of the present invention, which is defined solely bythe claims/items.

All publications and patents cited throughout the text of thisspecification (including all patents, patent applications, scientificpublications, manufacturer's specifications, instructions, etc.),whether supra or infra, are hereby incorporated by reference in theirentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material.

SUMMARY

The present invention is based on the surprising findings ofpolynucleotide sequences (“polynucleotides of the present invention”)encoding a protein which is involved in lipid metabolism (“helperprotein” of the present invention) whose expression, preferablyoverexpression led to an increase in the yield of protein of interest(POI). This disclosure provides methods and materials useful forimproving the yield of POI by engineering host cells such that they arecapable of overexpressing at lesst one, i.e., one or more proteins whichare involved in lipid metabolism. Preferred polynucleotides encode ahelper protein (involved in lipid metabolism) comprising an amino acidsequence as shown in any one of SEQ ID NOs: 1-15, such as SEQ ID NO:1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or a functionalhomologue thereof, wherein the functional homologue has at least 30%sequence identity to an amino acid sequence as shown in any one of SEQID NOs: 1-15, respectively.

The present invention is also based on the surprising findings ofpolynucleotide sequences (“polynucleotides of the present invention”)encoding a protein which is involved in lipid metabolism (“helperprotein of the present invention”) whose underexpression led to anincrease in the yield of protein of interest (POI). Such helper proteinsare sometimes also referred to herein as “KO protein” or “KO helperprotein”. Accordingly, the term “helper protein” or “helper gene” or“helper factor” etc. also includes “KO helper protein” or “KO helpergene” or “KO helper factor”, if this technically makes sense (i.e. ifunderexpression results in an increase in the yield of POI), even if inthe text of the present application the term used is only “helperprotein” or “helper gene” or “helper factor” etc. This disclosure alsoprovides methods and materials useful for improving the yield of POI byengineering host cells such that they are capable of underexpressing atleast one, i.e., one or more proteins which are involved in lipidmetabolism. Preferred polynucleotides encode a KO protein (involved inlipid metabolism) comprising an amino acid sequence as shown in SEQ IDNO: 16 or 17, or a functional homologue thereof, wherein the functionalhomologue has at least 30% sequence identity to an amino acid sequenceas shown in any one of SEQ ID NOs: 16 or 17, respectively.

The findings of the present inventors are rather surprising, since lipidmetabolism was to the best of one's knowledge up to the presentinvention not brought in connection with increasing the yield of aprotein of interest in a eukaryotic host cell, particularly in a fungalhost cell. Without being bound by theory, modification of lipidmetabolism modifies biomembrane lipid composition in a eukaryotic hostcell, particularly in a fungal host cell, thereby positively affectingrecombinant protein production.

It is assumed that overexpression of at least one protein involved inlipid metabolism, preferably of at least one of the helper proteinsdescribed herein changes the lipid composition of a membrane, inparticular the cellular membrane or the endoplasmatic reticulummembrane, of a host cell as described herein which results in increasingthe yield of a protein of interest.

Specifically, it is assumed that overexpression of at least one proteininvolved in lipid metabolism, preferably of at least one of the helperproteins described herein, alters the molecular species pattern ofsphingolipids, preferably by increasing the amount of C26 fatty acylmoieties of ceramides and/or inositol-containing phosphorylceramides(such as inositolphosphorylceramide (IPC),mannosyl-inositolphosphorylceramide (MIPC),mannosyl-diinositol-phosphorylceramide (M(IP)2C)) and/or decreasing theamount of C24 fatty acyl moieties of ceramides and/orinositol-containing phosphorylceramides (such asinositolphosphorylceramide (IPC), mannosyl-inositolphosphorylceramide(MIPC), mannosyl-diinositol-phosphorylceramide M(IP)2C) results inincreasing the yield of a protein of interest.

Preferably, the relative amount of fatty acyls of a chain length of 26carbons (C26) incorporated in ceramides, e.g. IPC, MIPC and M(IP)2C isincreased by at least 100%, whereas the relative amount of fatty acylsof a chain length of 24 carbons (C24) are decreased by at least 70%.

Preferably, overexpression of at least one protein involved in lipidmetabolism, preferably of at least one of the helper proteins describedherein, leads to an altered molecular species pattern of mostsphingolipids, that is fatty acyl moieties of ceramides and IPC-classesinositol-containing phosphorylceramides (IPC, MIPC, M(IP)2C)preferentially contain C26 instead of C24. The amount of C26 isdepending on the kind of sphingolipid (ceramides, IPC, MIPC and M(IP)2C)enhanced at least by 100% compared to the empty vector.

It is also assumed that overexpression of at least one protein involvedin lipid metabolism, preferably of at least one of the helper proteinsdescribed herein, reduces the amount of IPC and MIPC (of approximately230%) and/or increases the formation of the mature form ofinositol-containing phosphorylceramides, M(IP)2C by at least 6-fold(600%) which results in increasing the yield of a protein of interest

It is further assumed that underexpression of at least one proteininvolved in lipid metabolism, preferably of at least one of the helperproteins—here a KO helper protein—described herein depletes thenon-polar storage lipid triacylglycerol (TG) which results in increasingthe yield of a protein of interest.

The term “yield” refers to the amount of POI or model protein(s) asdescribed herein, in particular SDZ-Fab (SEQ ID NO: 37 for heavy chainand SEQ ID NO: 38 for light chain) and HyHEL-Fab (SEQ ID NO: 39 forheavy chain and SEQ ID NO: 40 for light chain), respectively, which is,for example, harvested from the engineered host cell, and increasedyields can be due to increased amounts of production or secretion of thePOI by the host cell. Yield may be presented by mg POI/g biomass(measured as dry cell weight or wet cell weight) of a host cell. Theterm “titer” when used herein refers similarly to the amount of producedPOI or model protein, presented as mg POI/L culture supernatant. Anincrease in yield can be determined when the yield obtained from anengineered host cell is compared to the yield obtained from a host cellprior to engineering, i.e., from a non-engineered host cell. Preferably,“yield” when used herein in the context of a model protein as describedherein, is determined as described in Example 4c. Accordingly, the“yield” when used herein in the context of a model protein as describedherein is also referred to as “Fab yield” or “Fab titer”. A Fab titer isgiven as mg/L and a Fab yield as mg/g biomass (measured as dry cellweight or wet cell weight). SDZ-Fab and HyHEL-Fab are encoded by thenucleotide sequences shown in SEQ ID NOs: 41 and 42 and SEQ ID NOs: 43and 44, respectively.

Briefly, P. pastoris strains CBS7435mut^(S) pPM2d_pAOX HyHEL and/orCBS7435mut^(S) pPM2d_pAOX SDZ (see Example 1 for their generation) whichexpress the model protein HyHEL-Fab and SDZ-Fab, respectively, areengineered with a polynucleotide encoding a helper protein or functionalhomologue thereof as described herein. For co-overexpression, the geneencoding a helper protein is cloned under control of the P. pastoris GAPpromoter and transformed into the Fab producing strains as described inExample 3b and Example 4. A gene may also be under control of the P.pastoris AOX promoter. For underexpression the gene encoding a KOprotein or its functional homologue is knocked out from the genome ofthe Fab producing strain (see Example 5). Engineered cells are grown inYP-medium containing 10 g/L glycerol and 50 μg/mL Zeocin overnight at25° C. (see Example 4a). Aliquots of such a culture (corresponding to afinal OD600 of 2.0) are transferred to synthetic medium M2 containing 20g/L glucose and a glucose feed tablet (described in Example 4a) andincubated for 25 h at 25° C. Cultures are washed and resuspended insynthetic medium M2 and aliquots (corresponding to a final OD600 of 4.0)are transferred into synthetic medium M2 supplemented with 5 g/Lmethanol. Methanol (5 g/L) is added every 12 hours. After 48 h, cellsare harvested by centrifugation. Biomass is determined by measuring theweight of the cell pellet derived from 1 mL cell suspension. Thesupernatant is used for quantification of SDZ-Fab or HyHEL-Fab,respectively, by ELISA (described in Example 4c). Specifically, ananti-human IgG antibody (e.g. ab7497, Abcam) is used as coating antibodyand a e.g. goat anti-human anti-human IgG (Fab specific) antibody (e.g.Sigma A8542, alkaline phosphatase conjugated) is used as detectionantibody. Commercial Human Fab/Kappa, IgG fragment is used as standardwith a starting concentration of 100 ng/mL, supernatant samples arediluted accordingly. An increase in the yield may be determined based ona comparison of POI yield before and after the cell is engineered tooverexpress the polypeptide. A standard test involving model proteinsSDZ-Fab and/or HyHEL-Fab as shown in the example may be used todetermine the yield difference.

Accordingly, the present invention relates to one or more newlydiscovered polypeptides involved in lipid metabolism (herein alsoreferred to as “helper proteins”) and its or their use to increase POIyield. The present invention is based on, but not limited to, the helperproteins shown in any one of SEQ ID NOs: 1 to 17 or functionalhomologues thereof. The meaning of functional homologue is defined inthe latter part of the application. The nucleotide sequences of thehelper proteins of the present invention are listed respectively in SEQID NOs: 19 to 35, respectively. As used herein, such proteins arereferred to in the present invention interchangeably in plural orsingular forms, which however should be understood as in singular formunless expressly stated otherwise. A helper protein involved in lipidmetabolism comprising an amino acid sequence as shown in any one of SEQID NOs: 1-15 is preferably overexpressed in a eukaryotic host cell,while a helper protein involved in lipid metabolism comprising an aminoacid sequence as shown in SEQ ID NO: 16 or 17 is preferablyunderexpressed in a eukaryotic host cell.

The invention additionally relates to the polynucleotides encoding thehelper proteins (hereinafter referred to as “polynucleotides of thepresent invention” or “a polynucleotide of the present invention”) andtheir individual or combined use to increase POI yield. Thepolynucleotide(s) can be introduced into a host cell or, if alreadyexisting in the cell, manipulated in a way such that they areoverexpressed. The polynucleotide of the present invention used foroverexpression of a helper protein encodes any one of SEQ ID NOs: 1 to15, such as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 or functional homologues thereof. Examples of the polynucleotidesequences are as set forth in SEQ ID NOs: 19 to 33, such as SEQ ID NO:19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33. Apolynucleotide encoding a helper protein involved in lipid metabolismcomprising a nucleotide sequence as shown in any one of SEQ ID NOs:19-33 is preferably overexpressed in a eukaryotic host cell, while apolynucleotide encoding a helper protein involved in lipid metabolismcomprising a nucleotide sequence as shown in SEQ ID NO: 34 or 35 ispreferably underexpressed in a eukaryotic host cell.

Accordingly, the present invention provides a method of increasing theyield of a protein of interest in a eukaryotic host cell, comprisingoverexpressing in said host cell at least one polynucleotide encoding ahelper protein which is involved in lipid metabolism, thereby increasingthe yield of said protein of interest in comparison to a host cell whichdoes not overexpress a polynucleotide encoding a protein which isinvolved in lipid metabolism. Preferably, said helper protein which isinvolved in lipid metabolism is not a transcription factor. Preferably,said helper protein which is involved in lipid metabolism comprises anamino acid sequence as shown in any one of SEQ ID NOs: 1-15 such as SEQID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or afunctional homologue thereof, wherein the functional homologue has atleast 30% sequence identity to an amino acid sequence as shown in anyone of SEQ ID NOs: 1-15, respectively.

Preferably, in the context of overexpression of helper proteins, lipidmetabolism is sphingolipid biosynthesis, phospholipid biosynthesis,lipid transport, or ergosterol biosynthesis. Modifying lipid metabolismby overexpression of a helper protein of the present invention canmodify biomembrane lipid composition in a eukaryotic host cell,particularly in a fungal host cell, thereby positively affectingrecombinant protein production. Hence, when used herein, the term “lipidmetabolism” in the context of overexpression can be replaced by the term“modification of biomembrane lipid composition”, preferably modificationof biomembrane lipid composition in a eukaryotic host cells,particularly in a fungal host cell. Modification of biomembrane lipidcomposition may be affected by sphingolipid biosynthesis, phospholipidbiosynthesis, lipid transport, or ergosterol biosynthesis.

The present invention furthermore provides a method for increasing theyield of a protein of interest in a eukaryotic host cell comprising

-   -   engineering the host cell to overexpress a polynucleotide        encoding a protein which is involved in lipid metabolism and        which comprises an amino acid sequence as shown in any one of        SEQ ID NOs: 1-15, such as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9,        10, 11, 12, 13, 14, or 15, preferably as shown in any one of SEQ        ID NOs: 1-9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NOs: 12-15,        or a functional homologue thereof, wherein the functional        homologue has at least 30% sequence identity to an amino acid        sequence as shown in any one of SEQ ID NOs: 1-15;    -   engineering said host cell to comprise a heterologous        polynucleotide encoding said protein of interest,    -   culturing said host cell under suitable conditions to express        said protein of interest, and optionally    -   isolating said protein of interest from the cell culture.

The present invention moreover provides a method for increasing theyield of a protein of interest in a eukaryotic host cell comprising

-   -   providing the host cell engineered to overexpress a        polynucleotide encoding a protein which is involved in lipid        metabolism and which comprises an amino acid sequence as shown        in any one of SEQ ID NOs: 1-15, such as SEQ ID NO:1, 2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, preferably as shown in        any one of SEQ ID NOs: 1-9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ        ID NOs: 12-15, or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in any one of SEQ ID NOs: 1-15,        wherein said host cell comprises a heterologous polynucleotide        encoding said protein of interest;    -   culturing the host cell under suitable conditions to overexpress        the protein involved in lipid metabolism or functional homologue        thereof and express said protein of interest, and optionally    -   isolating said protein of interest from the cell culture.

In the context of methods for increasing the yield of a protein theorder of the “engineering to overexpress/underexpress a polynucleotideencoding a helper protein step” and “engineering to comprise aheterologous polynucleotide encoding the protein of interest step” canalternatively be reversed such that the “engineering to comprise aheterologous polynucleotide encoding the protein of interest step”precedes the “engineering to overexpress/underexpress a polynucleotideencoding a helper protein step”. Notably, as described herein, the yieldof a protein of interest is increased when a helper protein isoverexpressed and/or a KO protein is underexpressed.

The present invention also provides a recombinant eukaryotic host cellfor manufacturing a protein of interest, wherein the host cell isengineered to overexpress a polynucleotide encoding a protein which isinvolved in lipid metabolism.

Preferably, the present invention provides a recombinant host cell formanufacturing a protein of interest, wherein the host cell is engineeredto overexpress a polynucleotide encoding a helper protein comprising anyone of SEQ ID NOs: 1-15, such as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15, preferably any one of SEQ ID NOs: 1-9, SEQ IDNO: 10, SEQ ID NO: 11, or SEQ ID NOs: 12-15, or a functional homologuethereof, wherein the functional homologue has at least 30% sequenceidentity to an amino acid sequence as shown in any one of SEQ ID NOs:1-15, respectively.

In a preferred embodiment, the helper protein, preferably whenoverexpressed, may increase the yield of the model protein SDZ-Fab (SEQID NO: 37 for heavy chain and SEQ ID NO: 38 for light chain; FIG. 2 ) orHyHEL-Fab (SEQ ID NO: 39 for heavy chain and SEQ ID NO: 40 for lightchain; FIG. 2 ) in the host cell by at least 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or at least200%, and preferably by at least 10% compared to the host cell prior tobeing engineered to overexpress said helper protein. A host cell priorto engineering does not overexpress the helper protein of the presentinvention and after engineering is able to overexpress the helperprotein under suitable culturing conditions. It has been surprisinglyfound that exemplary recombinant cells described in the Examples wereall able to increase the yield of the model protein SDZ-Fab or HyHEL-Fabby at least 20% (1.2 fold change).

The present invention also relates to a method of increasing the yieldof a protein of interest in a eukaryotic host cell, comprisingunderexpressing in said host cell at least one polynucleotide encoding ahelper protein which is involved in lipid metabolism, thereby increasingthe yield of said protein of interest in comparison to said host cellwhich does not underexpress a polynucleotide encoding a protein which isinvolved in lipid metabolism. Preferably, said helper protein which isinvolved in lipid metabolism comprises an amino acid sequence as shownin SEQ ID NO: 16 or 17 or a functional homologue thereof, wherein thefunctional homologue has at least 30% sequence identity to an amino acidsequence as shown in SEQ ID NO: 16 or 17.

Accordingly, the present invention provides for a method of increasingthe yield of a protein of interest in a eukaryotic host cell, comprising

-   -   engineering the host cell to underexpress a polynucleotide        encoding a protein which is involved in lipid metabolism and        which comprises an amino acid sequence as shown in SEQ ID NO: 16        or 17, or a functional homologue thereof, wherein the functional        homologue has at least 30% sequence identity to an amino acid        sequence as shown in SEQ ID NO: 16 or 17,    -   engineering said host cell to comprise a heterologous        polynucleotide encoding said protein of interest,    -   culturing said host cell under suitable conditions to express        said protein of interest, and optionally    -   isolating said protein of interest from the cell culture.

Also, the present invention provides a method of increasing the yield ofa protein of interest in a eukaryotic host cell, comprising

-   -   providing the host cell engineered to underexpress a        polynucleotide encoding a protein which is involved in lipid        metabolism and which comprises an amino acid sequence as shown        in SEQ ID NO: 16 or 17, or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in SEQ ID NOs: 16 or        17, wherein said host cell comprises a heterologous        polynucleotide encoding said protein of interest;    -   culturing the host cell under suitable conditions to        underexpress the protein which is involved in lipid metabolism        or functional homologue thereof and express said protein of        interest, and optionally    -   isolating said protein of interest from the cell culture.

In the context of methods for increasing the yield of a protein theorder of the “engineering to overexpress/underexpress a polynucleotideencoding a helper protein step” and “engineering to comprise aheterologous polynucleotide encoding the protein of interest step” canalternatively be reversed such that the “engineering to comprise aheterologous polynucleotide encoding the protein of interest step”precedes the “engineering to overexpress/underexpress a polynucleotideencoding a helper protein step”. Notably, as described herein, the yieldof a protein of interest is increased when a helper protein isoverexpressed and/or a KO protein is underexpressed.

Also provided herein is a recombinant eukaryotic host cell formanufacturing a protein of interest, wherein the host cell is engineeredto underexpress a polynucleotide encoding a protein which is involved inlipid metabolism. Preferably, said protein involved in lipid metabolismcomprises an amino acid sequence as shown in SEQ ID NO: 16 or 17, or afunctional homologue thereof, wherein the functional homologue has atleast 30% sequence identity to an amino acid sequence as shown in SEQ IDNO: 16 or 17.

Preferably, in the context of underexpression of a helper protein, thelipid metabolism in which the helper protein is involved is lipidstorage. Preferably, in the context of underexpression of a helperprotein, the helper protein is not a transcription factor. Modifyinglipid metabolism by underexpression of a helper protein of the presentinvention can modify biomembrane lipid composition in a eukaryotic hostcell, particularly in a fungal host cell, thereby positively affectingrecombinant protein production. Hence, when used herein, the term “lipidmetabolism” in the context of underexpression can be replaced by theterm “modification of biomembrane lipid composition”, preferablymodification of biomembrane lipid composition in a eukaryotic hostcells, particularly in a fungal host cell. Modification of biomembranelipid composition may be affected by non-polar storage lipidbiosynthesis or phospholipid metabolism.

In a preferred embodiment, the helper protein, preferably whenunderexpressed, may increase the yield of the model protein SDZ-Fab (SEQID NO: 37 for heavy chain and SEQ ID NO: 38 for light chain) orHyHEL-Fab (SEQ ID NO: 39 for heavy chain and SEQ ID NO: 40 for lightchain) in the host cell by at least 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or at least 200%, andpreferably by at least 10% compared to the host cell prior to beingengineered to underexpress said helper protein. A host cell prior toengineering does not underexpress the helper protein of the presentinvention and after engineering is able to underexpress the helperprotein under suitable culturing conditions. It has been surprisinglyfound that exemplary recombinant cells described in the Examples wereall able to increase the yield of the model protein SDZ-Fab or HyHEL-Fabby at least 20% (1.2 fold change). In some instances, the yieldincreased by 80%, as shown in Example 6b.

The present invention also provides for uses of the host cell asdescribed herein for manufacturing a protein of interest (POI). The hostcells can be advantageously used for introducing polynucleotidesencoding one or more POI(s), and thereafter can be cultured undersuitable condition to express the POI, whereby a helper protein of thepresent invention is overexpressed or underexpressed.

An isolated polynucleotide encoding a helper protein involved in lipidmetabolism and comprising an amino acid sequence as shown in any one ofSEQ ID NOs: 1-17, such as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, or 17, or a functional homologue thereof, whereinthe functional homologue has at least 30% sequence identity to an aminoacid sequence as shown in any one of SEQ ID NOs: 1-17, respectively, isalso provided by the present invention. Preferably, an isolatedpolynucleotide encoding a helper protein involved in lipid metabolismand comprising an amino acid sequence as shown in any one of SEQ ID NOs:1-15, such as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,or 15, or a functional homologue thereof, wherein the functionalhomologue has at least 30% sequence identity to an amino acid sequenceas shown in any one of SEQ ID NOs: 1-15, respectively, is also providedby the present invention.

Likewise, an isolated polynucleotide encoding a helper protein andcomprising the nucleotide sequence as shown in any one of SEQ ID NOs:19-36 is also provided by the present invention. A polynucleotide thatis used for integration in a host cell or for manufacturing a protein ofinterest comprises preferably a nucleotide sequence as shown in any oneof SEQ ID NOs: 19-33 or 36. Similarly, an isolated helper protein asdescribed herein is used for manufacturing a protein of interest. Such apolynucleotide encodes a helper protein comprising an amino acidsequence as shown in any one of SEQ ID NOs: 1-15.

The present invention also relates to a composition comprising at least0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, of a protein of interest and apolynucleotide encoding a helper protein of the present invention,wherein said polynucleotide is operably linked with a heterologouspromoter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid and polynucleotide sequences of the helperproteins of the present invention.

FIG. 2 shows the amino acid and polynucleotide sequences of the heavychain and light chain of the model proteins SDZ-Fab and HyHEL-Fab,respectively and the S. cerevisiae alpha mating factor signal leadersequence. The underlined sequences parts represent leader sequences ofSEQ ID NO. 45 or 46 fused to the model protein sequence.

ITEMS OF THE INVENTION

-   1. A method of increasing the yield of a protein of interest in a    eukaryotic host cell, comprising overexpressing in said host cell at    least one polynucleotide encoding at least one protein which is    involved in lipid metabolism, thereby increasing the yield of said    protein of interest in comparison to a host cell which does not    overexpress a polynucleotide encoding a protein which is involved in    lipid metabolism.-   2. The method of item 1, wherein said protein which is involved in    lipid metabolism is involved in sphingolipid biosynthesis,    phospholipid biosynthesis, lipid transport, lipid storage,    ergosterol biosynthesis, fatty acid biosynthesis, phosphatidic acid    biosynthesis and/or phospholipid metabolic process.-   3. The method of item 1 or 2, wherein said protein which is involved    in lipid metabolism is not a transcription factor.-   4. The method of any one of items 1-3, wherein said at least one    protein which is involved in lipid metabolism comprises an amino    acid sequence as shown in any one of SEQ ID NOs: 1-15 or a    functional homologue thereof, wherein the functional homologue has    at least 30% sequence identity to an amino acid sequence as shown in    any one of SEQ ID NOs: 1-15.-   5. The method of any one of items 1-4, wherein said at least one    protein which is involved in sphingolipid biosynthesis comprises an    amino acid sequence as shown in any one of SEQ ID NOs: 1-9 or a    functional homologue thereof, wherein the functional homologue has    at least 30% sequence identity to an amino acid sequence as shown in    any one of SEQ ID NOs: 1-9.-   6. The method of any one of items 1-4, wherein said at least one    protein which is involved in phospholipid biosynthesis comprises an    amino acid sequence as shown in SEQ ID NO: 10 or a functional    homologue thereof, wherein the functional homologue has at least 30%    sequence identity to an amino acid sequence as shown in SEQ ID NO:    10.-   7. The method of any one of items 1-4, wherein said at least one    protein which is involved in lipid transport comprises an amino acid    sequence as shown in SEQ ID NO: 11 or a functional homologue    thereof, wherein the functional homologue has at least 30% sequence    identity to an amino acid sequence as shown in SEQ ID NO: 11.-   8. The method of any one of items 1-4, wherein said at least one    protein which is involved in ergosterol biosynthesis comprises an    amino acid sequence as shown in any one of SEQ ID NOs: 12-15 or a    functional homologue thereof, wherein the functional homologue has    at least 30% sequence identity to an amino acid sequence as shown in    any one of SEQ ID NOs: 12-15.-   9. The method of any one of items 1-4, wherein said protein which is    involved in lipid storage comprises an amino acid sequence as set    forth between PP7435_Chr4-0493 and PP7435_Chr4-0494 (ARV1) or a    functional homologue thereof, wherein the functional homologue has    at least 30% sequence identity to an amino acid sequence as as set    forth between PP7435_Chr4-0493 and PP7435_Chr4-0494.-   10. The method of any one of items 1-9 comprising:    -   engineering the host cell to overexpress a polynucleotide        encoding a protein which is involved in lipid metabolism and        which comprises an amino acid sequence as shown in any one of        SEQ ID NOs: 1-15, preferably as shown in any one of SEQ ID NOs:        1-9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NOs: 12-15, or a        functional homologue thereof, wherein the functional homologue        has at least 30% sequence identity to an amino acid sequence as        shown in any one of SEQ ID NOs: 1-1529;    -   engineering said host cell to comprise a heterologous        polynucleotide encoding a protein of interest,    -   culturing said host cell under suitable conditions to express        said protein of interest, and optionally    -   isolating said protein of interest from the cell culture.-   11. A method of manufacturing a protein of interest according to any    one of items 1-9 comprising:    -   providing the host cell engineered to overexpress at least one        polynucleotide encoding at least one protein which is involved        in lipid metabolism and which comprises an amino acid sequence        as shown in any one of SEQ ID NOs: 1-15, preferably as shown in        any one of SEQ ID NOs: 1-9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ        ID NOs: 12-15, or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in any one of SEQ ID NOs: 1-15,        wherein said host cell comprises a heterologous polynucleotide        encoding a protein of interest;    -   culturing the host cell under suitable conditions to overexpress        the protein involved in lipid metabolism or functional homologue        thereof and express said protein of interest, and optionally    -   isolating said protein of interest from the cell culture.-   12. The method of any one of items 1-11, wherein overexpression is    achieved by having 1, 2, 3, 4 or more copies of said polynucleotide    encoding a protein which is involved in lipid metabolism or    functional homologue thereof in said host cell.-   13. The method of any one of items 1-12, wherein said polynucleotide    encoding a protein which is involved in lipid metabolism or    functional homologue thereof is integrated into at least one    chromosome of said host cell.-   14. The method of item 13, wherein the integration is ectopically    and/or in the natural locus.-   15. The method of item 14, wherein the polynucleotide encoding a    protein which is involved in lipid metabolism or functional    homologue thereof is integrated in AOX1, GAP, ENO1, TEF, HIS4, TYR1,    HIS3, LEU2, URA3, LYS2, ADE2, TRP1, GAL1, or ADH1 locus of the host    cell genome.-   16. The method of any one of items 1-12, wherein the polynucleotide    encoding a protein which is involved in lipid metabolism or    functional homologue thereof is contained in a vector or plasmid.-   17. The method of item 15, wherein the vector is YIp type vector,    YEp type vector, YRp type vector, YCp type vector, pGPD-2, pAO815,    pGAPZ, pGAPZα, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, pPICZ, pPICZα,    pPIC3K, pHWO10, pPUZZLE, or 2 μm plasmid.-   18. The method of any one of items 1-17, wherein the overexpression    of the polynucleotide encoding a protein which is involved in lipid    metabolism or functional homologue thereof is achieved by using a    recombinant promoter which drives expression of said polynucleotide.-   19. The method of any one of items 1-17, wherein overexpression of    the polynucleotide encoding a protein which is involved in lipid    metabolism or functional homologue thereof is achieved by modifying    a regulatory sequence operably linked to the endogenous    polynucleotide encoding a protein which is involved in lipid    metabolism or functional homolog thereof.-   20. The method of item 18, wherein the promoter is PAOX1, PTPI,    PPGK, PGAPDH, PLAC, PGAL, PPGI, PGAP, PTEF, PENO1, PTPI, PRPS2,    PRPS7, PRPS31, PRPL1, PFLD, PICL, PTHI, PSSA1, PHSP90, PKAR2, PGND1,    PGPM1, PTKL1, PPIS1, PFET3, PFTR1, PPHO8, PNMT1, PMCM1, PUBI4,    PRAD2, PPET9, PFMD, PGAL1, PADH1, PADH2/GAP, PCUP1, or PMAL.-   21. The method of any one of items 1-17, wherein the overexpression    of the polynucleotide encoding a protein which is involved in lipid    metabolism or functional homologue thereof is achieved by using an    enhancer to enhance the promoter activity.-   22. The method of item 21, wherein the enhancer is the yeast    upstream activating sequence UAS/GAL.-   23. The method of any one of items 1-22, wherein the eukaryotic host    cell is a non-mammalian eukaryotic host cell.-   24. The method of item 23, wherein the non-mammalian eukaryotic host    cell is a fungal host cell.-   25. The method of item 24, wherein the fungal host cell is Pichia    pastoris, Hansenula polymorpha, Trichoderma reesei, Saccharomyces    cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia    methanolica, Candida boidinii, Komagataella sp., Aspergillus sp. or    Schizosaccharomyces pombe.-   26. The method of any one of items 1-25, wherein 1, 2, 3, 4, 5, 6,    7, 8 or more proteins involved in lipid metabolism selected from any    one of SEQ ID NOs: 1-15, PP7435_Chr3-0788 (SUR2), PP7435_Chr3-1005    (AUR1), PP7435_Chr4-0626 (IFA38), PP7435_Chr3-0669 (SCS7),    PP7435_Chr3-0636 (CRD1), PP7435_Chr3-0950 (SLC4), PP7435_Chr2-0585    (PIS1), PP7435_chr1-0934 (PRY2), PP7435_Chr4-0963 (FAD12) and    PP7435_Chr1-0794 (PSD1) or a functional homologues thereof are    overexpressed.-   27. The method of item 26, wherein the following at least one    protein involved in sphingolipid biosynthesis is overexpressed    -   (a) protein comprising the amino acid sequence as shown in SEQ        ID NO: 1 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 1;    -   (b) protein comprising the amino acid sequence as shown in SEQ        ID NO: 2 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 2;    -   (c) protein comprising the amino acid sequence as shown in SEQ        ID NO: 3 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 3;    -   (d) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 1 and 2 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NOs: 1 and 2,        respectively;    -   (e) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 1 and 3 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NOs: 1 and 3,        respectively;    -   (f) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 1, 3 and 4 or a functional homologue thereof, wherein        the functional homologue has at least 30% sequence identity to        an amino acid sequence as shown in SEQ ID NOs: 1, 3 and 4,        respectively;    -   (g) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 1, 3, 4 and 8 or a functional homologue thereof, wherein        the functional homologue has at least 30% sequence identity to        an amino acid sequence as shown in SEQ ID NOs: 1, 3, 4 and 8,        respectively;    -   (h) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 5 and 6 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NOs: 5 and 6,        respectively;    -   (i) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 1 and 7 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NOs: 1 and 7,        respectively;    -   (j) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 5, 6 and 9 or a functional homologue thereof, wherein        the functional homologue has at least 30% sequence identity to        an amino acid sequence as shown in SEQ ID NOs: 5, 6 and 9,        respectively;    -   (k) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 1, 5, 6 and 9 or a functional homologue thereof, wherein        the functional homologue has at least 30% sequence identity to        an amino acid sequence as shown in SEQ ID NOs: 1, 5, 6 and 9,        respectively;    -   (l) protein comprising the amino acid sequence as shown in SEQ        ID NO: 4 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 4;    -   (m) protein comprising the amino acid sequence as shown in SEQ        ID NO: 5 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 5;    -   (n) protein comprising the amino acid sequence as shown in SEQ        ID NO: 6 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 6;    -   (o) protein comprising the amino acid sequence as shown in SEQ        ID NO: 7 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 7    -   (p) protein comprising the amino acid sequence as shown in SEQ        ID NO: 8 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 8;    -   (q) protein comprising the amino acid sequence as shown in SEQ        ID NO: 9 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 9;    -   (r) protein comprising the amino acid sequence as shown in        PP7435_Chr3-0788 (SUR2) or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in PP7435_Chr3-0788;    -   (s) protein comprising the amino acid sequence as shown in        PP7435_Chr3-1005 (PHS1) or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in PP7435_Chr3-1005;    -   (t) protein comprising the amino acid sequence as shown in        PP7435_Chr2-0350 (AUR1) or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in PP7435_Chr2-0350;    -   (u) protein comprising the amino acid sequence as shown in        PP7435_Chr4-0626 (IFA38) or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in PP7435_Chr4-0626;    -   (v) protein comprising the amino acid sequence as shown in        PP7435_Chr3-0669 (SCS7) or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in PP7435_Chr3-0669,    -   (w) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 3 and 4 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NOs: 3 and 4,        respectively;    -   (x) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 3, 4 and 8 or a functional homologue thereof, wherein        the functional homologue has at least 30% sequence identity to        an amino acid sequence as shown in SEQ ID NOs: 3, 4 and 8,        respectively;    -   (y) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 1 and 5 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NOs: 1 and 5,        respectively;    -   (z) proteins comprising the amino acid sequences as shown in SEQ        ID NOs: 1, 5 and 6 or a functional homologue thereof, wherein        the functional homologue has at least 30% sequence identity to        an amino acid sequence as shown in SEQ ID NOs: 1, 5 and 6,        respectively;    -   (aa) proteins comprising the amino acid sequences as shown in        SEQ ID NO: 1 and PP7435_Chr3-0788 (SUR1) or a functional        homologue thereof, wherein the functional homologue has at least        30% sequence identity to an amino acid sequence as shown in SEQ        ID NO: 1 and PP7435_Chr3-0788, respectively;    -   (bb) proteins comprising the amino acid sequences as shown in        SEQ ID NO: 1 and PP7435_Chr3-1005 (PHS1) or a functional        homologue thereof, wherein the functional homologue has at least        30% sequence identity to an amino acid sequence as shown in SEQ        ID NO: 1 and PP7435_Chr3-1005, respectively;    -   (cc) proteins comprising the amino acid sequences as shown in        SEQ ID NO: 1 and 8 or a functional homologue thereof, wherein        the functional homologue has at least 30% sequence identity to        an amino acid sequence as shown in SEQ ID NO: 1 and 8,        respectively;    -   (dd) proteins comprising the amino acid sequences as shown in        SEQ ID NO: 1 and PP7435_Chr2-0350 (AUR1) or a functional        homologue thereof, wherein the functional homologue has at least        30% sequence identity to an amino acid sequence as shown in SEQ        ID NO: 1 and PP7435_Chr2-0350, respectively;    -   (ee) proteins comprising the amino acid sequences as shown in        SEQ ID NO: 1 and PP7435_Chr4-0626 (IFA38) or a functional        homologue thereof, wherein the functional homologue has at least        30% sequence identity to an amino acid sequence as shown in SEQ        ID NO: 1 and PP7435_Chr4-0626, respectively; or    -   (ff) proteins comprising the amino acid sequences as shown in        SEQ ID NO: 1 and PP7435_Chr3-0669 (SCS7) or a functional        homologue thereof, wherein the functional homologue has at least        30% sequence identity to an amino acid sequence as shown in SEQ        ID NO: 1 and PP7435_Chr3-0669, respectively.-   28. The method of item 26, wherein the following proteins involved    in phospholipid biosynthesis are overexpressed    -   (a) protein comprising the amino acid sequence as shown in SEQ        ID NO: 10 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 10;    -   (b) protein comprising the amino acid sequence as shown in        PP7435_Chr3-0636 (CRD1) or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in PP7435_Chr3-0636;    -   (c) protein comprising the amino acid sequence as shown in        PP7435_Chr3-0950 (SLC4) or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in PP7435_Chr3-0950;    -   (d) protein comprising the amino acid sequence as shown in        PP7435_Chr2-0585 (PIS1) or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in PP7435_Chr2-0585.-   29. The method of item 26, wherein the following proteins involved    in lipid transport are overexpressed    -   (a) protein comprising the amino acid sequence as shown in SEQ        ID NO: 11 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 11,    -   (b) protein comprising the amino acid sequence as shown in        PP7435_Chr1-0934 (PRY1) or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in PP7435_Chr1-0934;    -   (c) proteins comprising the amino acid sequence as shown in SEQ        ID NO: 11 and PP7435_Chr1-0934 (PRY1) or a functional homologue        thereof, wherein the functional homologue has at least 30%        sequence identity to an amino acid sequence as shown in SEQ ID        NO: 11 and PP7435_Chr1-0934, respectively.-   30. The method of item 26, wherein the following proteins involved    in fatty acid biosynthesis are overexpressed    -   (a) protein comprising the amino acid sequence as shown in        PP7435_Chr4-0963 (FAD12) or a functional homologue thereof,        wherein the functional homologue has at least 30% sequence        identity to an amino acid sequence as shown in PP7435_Chr4-0963.-   31. The method of item 26, wherein at least one protein involved in    sphingolipid biosynthesis and at least one protein involved in lipid    transport is overexpressed.-   32. The method of item 31, wherein the protein involved in    sphingolipid biosynthesis comprises an amino acid sequence as shown    in SEQ ID NO: 1 or a functional homologue thereof and the protein    involved in lipid transport comprises an amino acid sequence as    shown in SEQ ID NO: 11 or a functional homologue thereof, wherein    the functional homologue has at least 30% sequence identity to an    amino acid sequence as shown in SEQ ID NOs: 1 and 11, respectively.-   33. The method of item 26, wherein at least one protein involved in    phospholipid biosynthesis and at least one protein involved in lipid    transport is overexpressed.-   34. The method of item 33, wherein the protein involved in    phospholipid biosynthesis comprises an amino acid sequence as shown    in SEQ ID NO: 10 or PP7435_Chr1-0794 (PSD1) and the protein involved    in lipid transport comprises an amino acid sequence as shown in SEQ    ID NO: 11.-   35. The method of item 34, wherein the following proteins involved    in phospholipid biosynthesis or in in lipid transport are    overexpressed    -   a) proteins comprising the amino acid sequence as shown in SEQ        ID NO: 10 and 11 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 10 and 11,        respectively;    -   b) proteins comprising the amino acid sequence as shown in        PP7435_Chr1-0794 (PSD1) and SEQ ID NO: 11 or a functional        homologue thereof, wherein the functional homologue has at least        30% sequence identity to an amino acid sequence as shown in        PP7435_Chr1-0794 and SEQ ID NO: 11, respectively.-   36. The method of item 26, wherein at least one protein involved in    sphingolipid biosynthesis and at least one protein involved in    phospholipid biosynthesis is overexpressed.-   37. The method of item 36, wherein the protein involved in    sphingolipid biosynthesis comprises an amino acid sequence as shown    in SEQ ID NO: 1 and the protein involved in phospholipid    biosynthesis comprises an amino acid sequence as shown in    PP7435_Chr2-0585 (PIS1), PP7435_Chr3-0636 (CRD1) or SEQ ID NO: 10.-   38. The method of item 37, wherein the following proteins involved    in sphingolipid biosynthesis or in in phospholipid biosynthesis are    overexpressed    -   a) proteins comprising the amino acid sequence as shown in SEQ        ID NO: 1 and PP7435_Chr2-0585 (PIS1) or a functional homologue        thereof, wherein the functional homologue has at least 30%        sequence identity to an amino acid sequence as shown in SEQ ID        NO: 1 and PP7435_Chr2-0585, respectively;    -   b) proteins comprising the amino acid sequence as shown in SEQ        ID NO: 1 and 10 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 1 and 10,        respectively;    -   c) proteins comprising the amino acid sequence as shown in SEQ        ID NO: 1 and PP7435_Chr3-0636 (CRD1) or a functional homologue        thereof, wherein the functional homologue has at least 30%        sequence identity to an amino acid sequence as shown in SEQ ID        NO: 1 and PP7435_Chr3-0636, respectively.-   39. The method of item 26, wherein at least one protein involved in    sphingolipid biosynthesis and at least one protein involved in    phospholipid biosynthesis is overexpressed.-   40. The method of item 26, wherein at least one protein involved in    sphingolipid biosynthesis and at least one protein involved in    ergosterol biosynthesis is overexpressed.-   41. The method of item 40, wherein the protein involved in    sphingolipid biosynthesis comprises an amino acid sequence as shown    in SEQ ID NO: 1 and the protein involved in ergosterol biosynthesis    comprises an amino acid sequence as shown in SEQ ID NO: 12.-   42. The method of item 26, wherein at least one protein involved in    ergosterol biosynthesis and at least one protein involved in lipid    transport is overexpressed.-   43. The method of item 42, wherein the protein involved in    ergosterol biosynthesis comprises an amino acid sequence as shown in    SEQ ID NO: 12 or 13 and the protein involved in lipid transport    comprises an amino acid sequence as shown in SEQ ID NO: 11.-   44. The method of item 37, wherein the following proteins involved    in ergosterol biosynthesis or in in lipid transport are    overexpressed    -   a) proteins comprising the amino acid sequence as shown in SEQ        ID NO: 12 and 11 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 12 and 11,        respectively;    -   b) proteins comprising the amino acid sequence as shown in SEQ        ID NO: 13 and 11 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 13 and 11,        respectively.-   45. The method of item 26, wherein at least one protein involved in    sphingolipid biosynthesis and at least one protein involved in lipid    storage is overexpressed.-   46. The method of item 45, wherein the protein involved in    sphingolipid biosynthesis comprises an amino acid sequence as shown    in SEQ ID NO: 1 and the protein involved in lipid storage is ARV1,    which comprises an amino acid sequence located between    PP7435_Chr4-0493 and PP7435_Chr4-0493.-   47. The method of any one of items 1-46, further comprising    overexpressing a chaperone.-   48. The method of item 47, wherein the chaperone comprises an amino    acid sequence as shown in SEQ ID NO: 18 or a functional homologue    thereof, wherein the functional homologue has at least 30% sequence    identity to an amino acid sequence as shown in SEQ ID NO: 18.-   49. The method of item 47 or 48, wherein at least one protein    involved in sphingolipid biosynthesis and at least one chaperone is    overexpressed.-   50. The method of item 49, wherein the protein involved in    sphingolipid biosynthesis comprises an amino acid sequence as shown    in SEQ ID NO: 1 or a functional homologue thereof and the chaperone    comprises an amino acid sequence as shown in SEQ ID NO: 18 or a    functional homologue thereof, wherein the functional homologue has    at least 30% sequence identity to an amino acid sequence as shown in    SEQ ID NOs: 1 and 18, respectively.-   51. The method of item 47 or 48, wherein at least one protein    involved in ergosterol biosynthesis and at least one chaperone is    overexpressed.-   52. The method of item 51, wherein the protein involved in    ergosterol biosynthesis comprises an amino acid sequence as shown in    SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 or a functional    homologue thereof and the chaperone comprises an amino acid sequence    as shown in SEQ ID NO: 18 or a functional homologue thereof, wherein    the functional homologue has at least 30% sequence identity to an    amino acid sequence as shown in SEQ ID NOs: 13 and 18, respectively.-   53. The method of any one of items 1-52, wherein overexpression of    the at least one polynucleotide encoding a protein which is involved    in lipid metabolism changes the lipid composition of a membrane of    the host cell, wherein the membrane of the host cell is preferably    the cellular membrane or the endoplasmatic reticulum membrane.-   54. The method of any one of items 1-53, wherein said protein of    interest is a non-membrane protein of interest.-   55. The method of item 54, wherein said non-membrane protein of    interest is an enzyme, a therapeutic protein, a food additive or    feed additive.-   56. The method of item 55, wherein the therapeutic protein comprises    an antibody or an antibody fragment still having the activity of    binding its antigen.-   57. The method of any one of items 1-56, wherein said protein    involved in lipid metabolism or functional homologue thereof    increases the yield of the model protein HyHEL (SEQ ID NO: 39 for    heavy chain and SEQ ID NO: 40 for light chain) compared to the host    cell prior to engineering by at least 10%, at least 15%, at least    20%, at least 25%, at least 30%.-   58. A recombinant eukaryotic host cell for manufacturing a protein    of interest, wherein the host cell is engineered to overexpress at    least one polynucleotide encoding at least one protein which is    involved in lipid metabolism.-   59. The host cell of item 58, wherein said protein which is involved    in lipid metabolism is involved in sphingolipid biosynthesis,    phospholipid biosynthesis, lipid transport, ergosterol biosynthesis,    fatty acid biosynthesis, phosphatidic acid biosynthesis and/or    phospholipid metabolic process.-   60. The host cell of item 58 or 59, wherein said protein which is    involved in lipid metabolism is not a transcription factor.-   61. The host cell of any one of items 58-59, wherein said protein    involved in lipid metabolism comprises an amino acid sequence as    shown in any one of SEQ ID NOs: 1-15, preferably as shown in any one    of SEQ ID NOs: 1-9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NOs:    12-15, or a functional homologue thereof, wherein the functional    homologue has at least 30% sequence identity to an amino acid    sequence as shown in any one of SEQ ID NOs: 1-15.-   62. The host cell of any one of items 58-61, wherein said protein    involved in lipid metabolism or said functional homologue thereof    increases the yield of the model protein HyHEL (SEQ ID NO: 39 for    heavy chain and SEQ ID NO: 40 for light chain) compared to the host    cell prior to engineering by at least 10%.-   63. The host cell of any one of items 58-62, wherein the    overexpression is achieved by having 1, 2, 3, 4 or more copies of    said polynucleotide encoding a protein involved in lipid metabolism    or functional homologue thereof in said host cell.-   64. The host cell of any one items 58-63, wherein said    polynucleotide encoding a protein which is involved in lipid    metabolism or functional homologue thereof is integrated in the    genome of said host cell.-   65. The host cell of item 64, wherein the integration is ectopically    and/or in the natural locus.-   66. The host cell of item 65, wherein at least one of the    polynucleotide encoding a protein which is involved in lipid    metabolism or functional homologue thereof is integrated in AOX1,    GAP, ENO1, TEF, HIS4, TYR1, HIS3, LEU2, URA3, LYS2, ADE2, TRP1,    GAL1, or ADH1 locus of the host cell genome.-   67. The host cell of any one of items 58-66, wherein the    polynucleotide encoding a protein which is involved in lipid    metabolism or functional homologue thereof is contained in a vector    or plasmid.-   68. The host cell of item 67, wherein the vector is YIp type vector,    YEp type vector, YRp type vector, YCp type vector, pGPD-2, pAO815,    pGAPZ, pGAPZα, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, pPICZ, pPICZα,    pPIC3K, pHWO10, pPUZZLE, or 2 μm plasmid.-   69. The host cell of any one of items 58-68, wherein the    overexpression of the polynucleotide encoding a protein which is    involved in lipid metabolism or functional homologue thereof is    achieved by using a recombinant promoter which drives expression of    said polynucleotide.-   70. The host cell of item 69, wherein the promoter is PAOX1, PTPI,    PPGK, PGAPDH, PLAC, PGAL, PPGI, PGAP, PTEF, PENO1, PTPI, PRPS2,    PRPS7, PRPS31, PRPL1, PFLD, PICL, PTHI, PSSA1, PHSP90, PKAR2, PGND1,    PGPM1, PTKL1, PPIS1, PFET3, PFTR1, PPHO8, PNMT1, PMCM1, PUBI4,    PRAD2, PPET9, PFMD, PGAL1, PADH1, PADH2/GAP, PCUP1, or PMAL.-   71. The host cell of item 69, wherein the overexpression of the    polynucleotide of the polynucleotide encoding a protein which is    involved in lipid metabolism or functional homologue thereof is    achieved by using an enhancer to enhance the promoter activity.-   72. The host cell of item 71, wherein the enhancer is the yeast    upstream activating sequence UAS/GAL.-   73. The host cell of any one of items 58-72, wherein the eukaryotic    host cell is a non-mammalian eukaryotic host cell.-   74. The host cell of any one of item 73, wherein the non-mammalian    eukaryotic host cell is a fungal host cell.-   75. The host cell of item 74, wherein the host cell is Pichia    pastoris, Hansenula polymorpha, Trichoderma reesei, Saccharomyces    cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia    methanolica, Candida boidinii, Komagataella sp., Aspergillus sp. and    Schizosaccharomyces pombe.-   76. The host cell of any one items 58-75, wherein 1, 2, 3, 4, 5, 6,    7, 8 or more of the proteins which are involved in lipid metabolism    are selected from any one of SEQ ID NOs: 1 to 15, PP7435_Chr3-0788    (SUR2), PP7435_Chr3-1005 (AUR1), PP7435_Chr4-0626 (IFA38),    PP7435_Chr3-0669 (SCS7), PP7435_Chr3-0636 (CRD1), PP7435_Chr3-0950    (SLC4), PP7435_Chr2-0585 (PIS1), PP7435_chr1-0934 (PRY2),    PP7435_Chr4-0963 (FAD12) and PP7435_Chr1-0794 (PSD1) or functional    homologues thereof are overexpressed.-   77. The host cell of item 76, wherein the proteins as defined in    items 27-46 are overexpressed.-   78. The host cell of any one of items 58-77, which is further    engineered to overexpress a polynucleotide encoding a chaperone.-   79. The host cell of item 78, wherein the chaperone comprises an    amino acid sequence as shown in SEQ ID NO: 18 or a functional    homologue thereof, wherein the functional homologue has at least 30%    sequence identity to an amino acid sequence as shown in SEQ ID NO:    18.-   80. The host cell of item 78 or 79, wherein the proteins as defined    in items 48-52 are overexpressed.-   81. The host cell of any one of items 58-80, comprising a    heterologous polynucleotide sequence encoding said protein of    interest.-   82. The host cell of item 81, wherein said protein of interest is a    non-membrane protein of interest.-   83. The host cell of item 82, wherein said non-membrane protein of    interest is an enzyme, a therapeutic protein, a food additive or    feed additive.-   84. The host cell of item 83, wherein the therapeutic protein    comprises an antibody or an antibody fragment still having the    activity of binding its antigen.-   85. The host cell of any one of items 58-84, wherein overexpression    is achieved by modifying a regulatory sequence operably linked to    the endogenous polynucleotide encoding the protein involved in lipid    metabolism or functional homolog thereof.-   86. The host cell of any one of items 58-85, wherein overexpression    of the at least one polynucleotide encoding a protein which is    involved in lipid metabolism changes the lipid composition of a    biomembrane of the host cell.-   87. Use of the host cell of any one of items 58-86 for manufacturing    a protein of interest.-   88. Use of the host cell according to item 87, wherein the protein    of interest is a non-membrane protein of interest.-   89. An isolated polynucleotide encoding a protein involved in lipid    metabolism and comprising an amino acid sequence as shown in any one    of SEQ ID NOs: 1-15 or a functional homologue thereof or an isolated    polynucleotide encoding a chaperone and comprising an amino acid    sequence as shown in SEQ ID NO: 18 or a functional homologue    thereof, wherein the functional homologue has at least 30% sequence    identity to an amino acid sequence as shown in any one of SEQ ID    NOs: 1-15 and 18, respectively.-   90. An isolated polynucleotide comprising the nucleotide sequence as    shown in any one of SEQ ID NOs: 19-33 and 36.-   91. Use of the isolated polynucleotide according to item 89 or 90    for integration in a host cell.-   92. Use of a polynucleotide according to item 89 or 90 for    manufacturing a protein of interest, preferably a non-membrane    protein of interest.-   93. Use of a polynucleotide according to item 89 or 90 for    manufacturing a host cell.-   94. An isolated polypeptide comprising an amino acid sequence having    at least 30% identity to an amino acid sequence shown in any one of    SEQ ID NOs: 1-15 and 18-   95. Use of the isolated polypeptide according to item 94 for    manufacturing a protein of interest, preferably a non-membrane    protein of interest.-   96. A composition comprising at least 0.25%, 0.5%, 1%, 2%, 3%, 4%,    5%, or 10%, of a protein of interest and a polynucleotide according    to item 86 or 87, wherein said polynucleotide is operably linked    with a heterologous promoter and wherein the protein of interest is    preferably a non-membrane protein of interest.-   97. A method of increasing the yield of a protein of interest in a    eukaryotic host cell, comprising underexpressing in said host cell    at least one polynucleotide encoding a protein which is involved in    lipid metabolism, thereby increasing the yield of said protein of    interest in comparison to said host cell which does not underexpress    a polynucleotide encoding a protein which is involved in lipid    metabolism.-   98. The method of item 97, wherein said protein which is involved in    lipid metabolism is involved in lipid storage.-   99. The method of item 97 or 98, wherein said protein which is    involved in lipid metabolism is not a transcription factor.-   100. The method of item 98 or 99, wherein said protein which is    involved in lipid storage comprises an amino acid sequence as shown    in any one of SEQ ID NOs: 16 or 17 or a functional homologue    thereof, wherein the functional homologue has at least 30% sequence    identity to an amino acid sequence as shown in SEQ ID NO: 16 or 17.-   101. The method of item 100, wherein the following proteins involved    in lipid storage are underexpressed    -   a) proteins comprising amino acid sequences as shown in SEQ ID        NOs: 16 and 17 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 16 and 17,        respectively;    -   b) protein comprising an amino acid sequence as shown in SEQ ID        NO: 16 or a functional homologue thereof, wherein the functional        homologue has at least 30% sequence identity to an amino acid        sequence as shown in SEQ ID NO: 16;    -   c) protein comprising an amino acid sequence as shown in SEQ ID        NO: 17 or a functional homologue thereof, wherein the functional        homologue has at least 30% sequence identity to an amino acid        sequence as shown in SEQ ID NO: 17.-   102. The method of any one of items 97-101, wherein the protein of    interest is a non-membrane protein of interest.-   103. The method of any one of items 97-102, wherein the eukaryotic    host cell is a non-mammalian eukaryotic host cell.-   104. The method of item 103, wherein the non-mammalian eukaryotic    host cell is a fungal host cell, wherein the fungal host cell is    preferably Pichia pastoris, Hansenula polymorpha, Trichoderma    reesei, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia    lipolytica, Pichia methanolica, Candida Komagataella sp.,    Aspergillus sp. and Schizosaccharomyces pombe, wherein the fungal    host cell is even more preferred Pichia pastoris.-   105. The method of any one of items 97-104 comprising:    -   engineering the host cell to underexpress a polynucleotide        encoding a protein which is involved in lipid metabolism and        which comprises an amino acid sequence as shown in SEQ ID NO: 16        or 17, or a functional homologue thereof, wherein the functional        homologue has at least 30% sequence identity to an amino acid        sequence as shown in SEQ ID NO: 16 or 17,    -   engineering said host cell to comprise a heterologous        polynucleotide encoding said protein of interest,    -   culturing said host cell under suitable conditions to express        said protein of interest, and optionally    -   isolating said protein of interest from the cell culture.-   106. A method of manufacturing a protein of interest according to    any one of items 97-104 comprising:    -   providing the host cell engineered to underexpress a        polynucleotide encoding a protein which is involved in lipid and        which comprises an amino acid sequence as shown in SEQ ID NO: 16        or 17, or a functional homologue thereof, wherein the functional        homologue has at least 30% sequence identity to an amino acid        sequence as shown in SEQ ID NO: 16 or 17, wherein said host cell        comprises a heterologous polynucleotide encoding said protein of        interest;    -   culturing the host cell under suitable conditions to        underexpress the protein which is involved in lipid metabolism        or functional homologue thereof and express said protein of        interest, and optionally    -   isolating said protein of interest from the cell culture.-   107. The method of any one of items 97-106, wherein the following    proteins involved in lipid metabolism are underexpressed    -   a) proteins comprising the amino acid sequence as shown in SEQ        ID NOs: 16 and 17 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NOs: 16 and 17,        respectively.-   108. The method of any one of items 97-106, comprising    overexpressing in said host cell at least one polynucleotide    encoding a protein which is involved in lipid metabolism.-   109. The method of item 108, wherein said protein which is    overexpressed and involved in lipid metabolism is involved in lipid    storage, sphingolipid biosynthesis, phospholipid biosynthesis,    phospholipid metabolic process, or phosphatidic acid biosynthesis.-   110. The method of item 109, wherein at least one protein involved    in sphingolipid biosynthesis is overexpressed and at least two    proteins involved in lipid storage are underexpressed.-   111. The method of item 110, wherein the following protein involved    in sphingolipid biosynthesis is overexpressed and the following    proteins involved in lipid storage are underexpressed    -   a) protein comprising the amino acid sequence as shown in SEQ ID        NO: 1 or a functional homologue thereof is overexpressed and        proteins comprising the amino acid sequences as shown in SEQ ID        NO: 16 and 17 or functional homologues thereof are        underexpressed, wherein the functional homologue has at least        30% sequence identity to an amino acid sequence as shown in SEQ        ID NO: 1, 16 and 17, respectively.-   112. The method of item 109, wherein at least one protein involved    in phospholipid biosynthesis is overexpressed and at least two    proteins involved in lipid storage are underexpressed.-   113. The method of item 112, wherein the protein involved in    phospholipid biosynthesis comprises an amino acid sequence as shown    in PP7435_Chr3-0950 (SLC4), PP7435_Chr1-0160 (SLC1),    PP7435_CHR1-0078 (GPT2) or SEQ ID NO: 10 and the proteins involved    in lipid storage comprise amino acid sequences as shown in SEQ ID    NO: 16 and 17.-   114. The method of item 113, wherein the following protein involved    in phospholipid biosynthesis is overexpressed and the following    proteins involved in lipid storage are underexpressed    -   a) protein comprising the amino acid sequence as shown in        PP7435_Chr3-0950 (SLC4) or a functional homologue thereof is        overexpressed and proteins comprising the amino acid sequences        as shown in SEQ ID NO: 16 and 17 or functional homologues        thereof are underexpressed, wherein the functional homologue has        at least 30% sequence identity to an amino acid sequence as        shown in PP7435_Chr3-0950, SEQ ID NO: 16 and 17, respectively;    -   b) protein comprising the amino acid sequence as shown in        PP7435_Chr1-0160 (SLC1) or a functional homologue thereof is        overexpressed and proteins comprising the amino acid sequences        as shown in SEQ ID NO: 16 and 17 or functional homologues        thereof are underexpressed, wherein the functional homologue has        at least 30% sequence identity to an amino acid sequence as        shown in PP7435_Chr1-0160, SEQ ID NO: 16 and 17, respectively;    -   c) protein comprising the amino acid sequence as shown in        PP7435_CHR1-0078 (GPT2) or a functional homologue thereof is        overexpressed and proteins comprising the amino acid sequences        as shown in SEQ ID NO: 16 and 17 or functional homologues        thereof are underexpressed, wherein the functional homologue has        at least 30% sequence identity to an amino acid sequence as        shown in PP7435 CHR1-0078, SEQ ID NO: 16 and 17, respectively;    -   d) protein comprising the amino acid sequence as shown in SEQ ID        NO: 10 or a functional homologue thereof is overexpressed and        proteins comprising the amino acid sequences as shown in SEQ ID        NO: 16 and 17 or functional homologues thereof are        underexpressed, wherein the functional homologue has at least        30% sequence identity to an amino acid sequence as shown in SEQ        ID NO: 10, 16 and 17, respectively.-   115. The method of item 109, wherein at least one protein involved    in phospholipid metabolic process is overexpressed and at least two    proteins involved in lipid storage are underexpressed.-   116. The method of item 115, wherein the protein involved in    phospholipid metabolic process is overexpressed and comprises an    amino acid sequence as shown in PP7435_Chr2-0045 (CDS1) or a    functional homologue thereof and the proteins involved in lipid    storage are underexpressed and comprise amino acid sequences as    shown in SEQ ID NO: 16 or 17 or a functional homologue thereof,    wherein the functional homologue has at least 30% sequence identity    to an amino acid sequence as shown in PP7435_Chr2-0045, SEQ ID NOs:    16 and 17, respectively.-   117. The method of item 116, wherein at least one protein involved    in phosphatidic acid biosynthesis is overexpressed and at least two    proteins involved in lipid storage are underexpressed.-   118. The method of item 117, wherein the protein involved in    phosphatidic acid biosynthesis is overexpressed and comprises an    amino acid sequence as shown in PP7435_Chr3-1169 (DGK1) or a    functional homologue thereof and the proteins involved in lipid    storage are underexpressed and comprise amino acid sequences as    shown in SEQ ID NO: 16 or 17 or a functional homologue thereof,    wherein the functional homologue has at least 30% sequence identity    to an amino acid sequence as shown in PP7435_Chr3-1169, SEQ ID NOs:    16 and 17, respectively.-   119. The method of item 109, wherein at least one protein involved    in lipid storage is overexpressed and at least two proteins involved    in lipid storage are underexpressed.-   120. The method of item 119, wherein the protein involved in lipid    storage which is overexpressed comprises an amino acid sequence as    shown in PP7435_Chr3-0741 (ARE2) or a functional homologue thereof    and the proteins involved in lipid storage which underexpressed    comprise amino acid sequences as shown in SEQ ID NO: 16 or 17 or a    functional homologue thereof, wherein the functional homologue has    at least 30% sequence identity to an amino acid sequence as shown in    PP7435_Chr3-0741, SEQ ID NOs: 16 and 17, respectively.-   121. A recombinant eukaryotic host cell for manufacturing a protein    of interest, wherein the host cell is engineered to underexpress at    least one polynucleotide encoding a protein which is involved in    lipid metabolism, wherein the protein of interest is preferably a    non-membrane protein.-   122. The host cell of item 121, wherein said protein involved in    lipid metabolism is involved in lipid storage and comprises an amino    acid sequence as shown in any one of SEQ ID NOs: 16 or 17, or a    functional homologue thereof, wherein the functional homologue has    at least 30% sequence identity to an amino acid sequence as shown in    any one of SEQ ID NOs: 16 or 17.-   123. The host cell of item 121 or 122, wherein the following    proteins involved in lipid metabolism are underexpressed    -   a) proteins comprising the amino acid sequence as shown in SEQ        ID NOs: 16 and 17 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NOs: 16 and 17,        respectively;    -   b) protein comprising the amino acid sequence as shown in SEQ ID        NO: 16 or a functional homologue thereof, wherein the functional        homologue has at least 30% sequence identity to an amino acid        sequence as shown in SEQ ID NO: 16;    -   c) protein comprising the amino acid sequence as shown in SEQ ID        NO: 17 or a functional homologue thereof, wherein the functional        homologue has at least 30% sequence identity to an amino acid        sequence as shown in SEQ ID NO: 17.-   124. The host cell of any one of items 121 to 123, wherein at least    one polynucleotide encoding a protein which is involved in lipid    metabolism is overexpressed as defined in items 109-120.-   125. The host cell of any one of items 121 to 124, wherein said    protein involved in lipid metabolism or said functional homologue    thereof increases the yield of the model protein HyHEL (SEQ ID NO:    39 for heavy chain and SEQ ID NO: 40 for light chain) compared to    the host cell prior to engineering by at least 10%.-   126. A method of increasing the yield of a protein of interest in a    eukaryotic host cell by changing the lipid composition of a membrane    of the host cell, wherein the membrane of the host cell is    preferably the cellular membrane or the endoplasmatic reticulum    membrane.-   127. The method of item 126, wherein the lipid composition of a    membrane of said host cell is changed by overexpression of at least    one protein involved in lipid metabolism, preferably by one of the    helper proteins described herein.-   128. A method of increasing the yield of a protein of interest in a    eukaryotic host cell by altering the molecular species pattern of    sphingolipids, preferably by increasing the amount of C26 fatty acyl    moieties of ceramides and/or inositol-containing    phosphorylcerarmides (inositolphosphorylceramide,    mannosyl-inositolphosphorylceramide,    mannosyl-diinositol-phosphorylceramide) and/or decreasing the amount    of C24 fatty acyl moieties of ceramides and/or inositol-containing    phosphorylceramides (inositolphosphorylceramide,    mannosyl-inositolphosphorylceramide,    mannosyl-diinositol-phosphorylceramide).-   129. The method of item 128, wherein the molecular species pattern    of sphingolipids is altered by overexpression of at least one    protein involved in lipid metabolism, preferably by one of the    helper proteins described herein.-   130. The method of item 128 or 129, wherein the relative amount of    fatty acyls of a chain length of 26 carbons (C26) incorporated in    ceramides, IPC, MIPC and M(IP)2C is increased by at least 100%,    whereas the the relative amount of fatty acyls of a chain length of    24 carbons (C24) are decreased by at least 70%.-   131. A method of increasing the yield of a protein of interest in a    eukaryotic host cell by reducing the amount of IPC and MIPC (of    approximately 30%) and/or by increasing the formation of the mature    form of inositol-containing phosphorylceramides, M(IP)2C by at least    6-fold (600%).-   132. The method of item 131, wherein said reduction and/or increment    is achieved by overexpression of at least one protein involved in    lipid metabolism, preferably by one of the helper proteins described    herein-   133. The method of item 131 or 132, wherein the relative    distribution of sphingolipids is affected as follows: 6-fold (600%)    increase in M(IP)2C and approximately 30% decrease in the relative    distribution of IPC and MIPC.-   134. A method of increasing the yield of a protein of interest in a    eukaryotic host cell by depleting the cell of the non-polar storage    lipid triacylglycerol (TG).-   135. The method of item 134, wherein said depletion is achieved by    underexpression of at least one protein involved in lipid    metabolism, preferably by one of the helper proteins described    herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is partly based on the surprising finding of theexpression of the helper proteins involved in lipid metabolism asdescribed herein, which were found to increase the yield of a protein ofinterest, if overexpressed (helper protein comprising an amino acidsequence shown in SEQ ID Nos: 1-15) or underexpressed (KO helper proteincomprising an amino acid sequence shown in SEQ ID Nos: 16 and 17). Theamino acid sequence and nucleotide sequences of each helper protein andits corresponding gene identifier which is sometimes used herein,particularly in the Examples are listed in Table 1 designated as “OE”(overexpressed helper proteins) and as “KO” (underexpressed helperproteins), below. Combinations of overexpressed and underexpressedhelper proteins are described herein and in detail in Example 6.

TABLE 1 Gene identities of Pichia pastoris sequences are retrieved fromSturmberger et al. . [J. Biotechnol. (2016). 235(4): 121-131)] and geneidentities of Saccharomyces cerevisiae sequences are retrieved fromCherry J. M. et al. [Nucleic Acids Res. (2012) 40 (Database issueD700-5)]._Gene ontology terms are defined as follows: “Sphingolipid”means sphingolipid biosynthesis including fatty acid elongation;“Transport” means lipid transport; “Phospholipid” means.phospholipidbiosynthesis; “Sterol” meansergosterol biosynthesis and “Storage” meanslipid storage. Gene accession numbers were retrieved from the EuropeanNucleotide Archive (ENA) and Protein accession numbers were retrievedfrom UniProtKB. SEQ SEQ IN ID Gene Gene Protein OE or Gene Identifier(ORF name NO NO Ontology accession accession KO Gene Pichia pastorisCBS7435) (AA) (DNA) Term number number OE ELO3 PP7435_chr3-0987 1 19Sphingolipid CCA39933.1 F2QX05 OE ELO2 PP7435_Chr3-0603 2 20Sphingolipid CCA39561.1 F2QVY3 OE LAG1 PP7435_Chr1-0676 3 21Sphingolipid CCA36821.1 F2QP79 OE LAC1 PP7435_Chr2-0202 4 22Sphingolipid CCA37899.1 F2QR71 OE LCB1 PP7435_Chr1-1525 5 23Sphingolipid CCA37636.1 F2QQF8 OE LCB2 PP7435_Chr3-0462 6 24Sphingolipid CCA39423.1 F2QVJ5 OE TSC13 PP7435_Chr4-0176 7 25Sphingolipid CCA40351.1 F2QY73 SEQ SEQ Gene Identifier (ORF name IN INOE or Saccharomyces cerevisiae NO NO Gene Ontology KO Gene S288C) (AA)(DNA) Term OE LIP1_(Sc) YMR298W 8 26 Sphingolipid CAA56807.1 Q03579 OETSC3_(Sc) YBR058C-A 9 27 Sphingolipid DAA07178.2 Q3E790 SEQ SEQ IN IN OEor Gene Identifier (ORF name NO NO Gene Ontology KO Gene Pichia pastorisCBS7435) (AA) (DNA) Term OE PAH1 PP7435_chr3-0694 10 28 PhospholipidCCA39650.1 F2QW72 OE PRY1 PP7435_chr3-1160 11 29 Transport CCA40103.1F2QXH5 OE ERG11 PP7435_chr3-0214 12 30 Sterol CCA39186.1 F2QUV8 OE HMG1PP7435_chr2-0242 13 31 Sterol CCA37938.1 F2QRB0 OE t2HMG1^(§)PP7435_chr2-0242_truncated 14 32 Sterol CCA37938.1 F2QRB0 OE t1HMG1^(§)PP7435_chr2-0242_truncated 15 33 Sterol CCA37938.1 F2QRB0 KO DGA1PP7435_chr3-1009 16 34 Storage CCA39955.1 F2QX27 KO LRO1PP7435_chr2-0587 17 35 Storage CCA38274.1 F2QS96 OE KAR2PP7435_Chr2-1167 18 36 Chaperone CAY68747.1* C4QZS3* ^(§)DNA-Sequencesof HMG1 in the table above were next to the full-length sequence used astwo truncated variants (t1, t2) which were depleted of their membraneanchoring domains that is depletion of 1449 nucleotides at the 5′-endfor variant t1HMG1 or 1543 nucleotides in case of t2HMG1 (except theirstarting ATG-codon). *KAR2 sequences (nucleotide as well as amino acid)are only annotated for P. pastoris strain GS115 which is almostidentical to CBS7435. Therefore, Gene/Protein accession numbers for KAR2are listed for the sequeneces annotated in the background of P. pastorisstrain GS115. Notably, for avoidance of doubt, the amino acid ornucleotide sequences referred to in the SEQ ID NOs. supersede any othersource of such sequences.

A “helper protein” or “helper protein of the present invention” as usedin the present invention means a protein which is involved in lipidmetabolism and which increases the yield of a model protein as describedherein or of a protein of interest (POI), preferably when being eitheroverexpressed in case of the helper proteins shown in SEQ ID NOs: 1-15,such as SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15,or when being underexpressed in case of the helper proteins shown in SEQID NOs: 16 or 17, in a host cell which also expresses the protein ofinterest. Additional helper proteins of the present invention that areinvolved in lipid metabolism and which enhance the yield of a modelprotein when being overexpressed are shown in PP7435_Chr3-0788 (SUR2),PP7435_Chr3-1005 (PHS1), PP7435_Chr2-0350 (AUR1), PP7435_Chr4-0626(IFA38), PP7435_Chr3-0669 (SCS7), PP7435_Chr3-0636 (CRD1),PP7435_Chr3-0950 (SLC4), PP7435_Chr2-0585 (PIS1), PP7435_chr1-0934(PRY2), ARV1 (ORF not annotated; located between PP7435_Chr4-0493/0494),PP7435_Chr3-0741 (ARE2), PP7435_Chr4-0963 (FAD12), PP7435_Chr1-0794(PSD1), PP7435_Chr1-0160 (SLC1), PP7435_CHR1-0078 (GPT2)PP7435_Chr3-1169 (DGK1) and PP7435_Chr2-0045 (CDS1) or functionalhomologues thereof. This term should be understood broadly and shouldnot be limited to, e.g. chaperones or chaperone-like proteins. As willappear evident from the present disclosure, helper proteins of thepresent invention are varied in their functions, however are allinvolved in certain classes of lipid metabolism. A preferred helperprotein of the present invention comprises the amino acid sequences ofany one of SEQ ID NOs: 1 to 17 or a functional homologue thereof. Apreferred helper protein of the present invention can be encoded by thenucleotide sequences of any one of SEQ ID NOs: 19 to 36 or variantsthereof encoding a functional homologue of SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15, respectively. For the purpose of thepresent invention, the term “helper protein” is also meant to encompassfunctional homologues of a helper protein as shown in any one of SEQ IDNOs: 1 to 17, respectively. The invention provides an isolatedpolynucleotide sequence encoding a helper protein comprising an aminoacid sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, or 15, or a functional homologue thereof, wherein thefunctional homologue has at least 30% sequence identity to an amino acidsequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15. The invention provides an isolated polynucleotidesequence encoding a helper protein comprising an amino acid sequence asshown in PP7435_Chr3-0788 (SUR2), PP7435_Chr3-1005 (PHS1),PP7435_Chr2-0350 (AUR1), PP7435_Chr4-0626 (IFA38), PP7435_Chr3-0669(SCS7), PP7435_Chr3-0636 (CRD1), PP7435_Chr3-0950 (SLC4),PP7435_Chr2-0585 (PIS1), PP7435_chr1-0934 (PRY2), ARV1 (ORF notannotated; located between PP7435_Chr4-0493/0494), PP7435_Chr3-0741(ARE2), PP7435_Chr4-0963 (FAD12), PP7435_Chr1-0794 (PSD1),PP7435_Chr1-0160 (SLC1), PP7435_CHR1-0078 (GPT2) PP7435_Chr3-1169 (DGK1)and PP7435_Chr2-0045 (CDS1) or a functional homologue thereof, whereinthe functional homologue has at least 30% sequence identity to an aminoacid sequence as shown in PP7435_Chr3-0788 (SUR2), PP7435_Chr3-1005(PHS1), PP7435_Chr2-0350 (AUR1), PP7435_Chr4-0626 (IFA38),PP7435_Chr3-0669 (SCS7), PP7435_Chr3-0636 (CRD1), PP7435_Chr3-0950(SLC4), PP7435_Chr2-0585 (PIS1), PP7435_chr1-0934 (PRY2), ARV1 (ORF notannotated; located between PP7435_Chr4-0493/0494), PP7435_Chr3-0741(ARE2), PP7435_Chr4-0963 (FAD12), PP7435_Chr1-0794 (PSD1),PP7435_Chr1-0160 (SLC1), PP7435_CHR1-0078 (GPT2) PP7435_Chr3-1169 (DGK1)and PP7435_Chr2-0045 (CDS1).

The term “involved in lipid metabolism” means that a helper protein hasfunction/activity in lipid metabolism of a eukaryotic cell. Lipidmetabolism includes, among others, functions such as lipid synthesis,lipid degradation, lipid transport (mediated via proteins, vesicles ordirect membrane contact with the purpose of bringing lipids from theirsite of synthesis to their site of destination e.g. incorporation intobiomembranes; or the transport of excess or malfunctioning lipids to thecell exterior), lipid storage (storage of lipids in biologically inertforms as steryl esters or triacylglycerols to avoid lipotoxicity causedby excess amounts of free sterols or fatty acids; lipid storage occursin specialized organelles the so-called lipid droplets), lipidmodification, lipid stability, lipid-lipid interaction, fatty acidbiosynthesis and modification, sphingolipid biosynthesis, phospholipidbiosynthesis, ergosterol biosynthesis, altering the molecular speciespattern of highly abundant sphingolipids, that is fatty acyl moieties ofceramides (ceramides and hexosyl-ceramides) and inositol-containingphosphorylceramides (IPC . . . inositolphosphorylceramide, MIPC . . .Mannosyl-inositolphosphorylceramide, M(IP)₂C . . .Mannosyl-diinositol-phosphorylceramide) preferentially contain C26 (26carbons in the fatty acyl chain) instead of C24 (24 carbons in the fattyacyl chain); altering the sphingolipid distribution pattern towardshigher occurrence of the mature form of complex sphingolipids that ismore M(IP)2C which comes at the expense of IPC, depleting cells oftriacylglycerols that is the major storage lipid of P. pastoris.Preferably, said helper protein which is involved in lipid metabolism isnot a transcription factor. Accordingly, a helper protein that isinvolved in lipid metabolism, either when it is over- or underexpressedas described herein, can modify biomembrane lipid composition in aeukaryotic host cell, particularly in a fungal host cell, therebypositively affecting recombinant protein production. The term“biomembrane” as used herein includes but is not limited to cellmembranes and endoplasmatic reticulum membrane, vesicle membrane, golgimembrane, etc. Hence, when used herein, the term “lipid metabolism” inthe context of over- and underexpression of a helper protein,respectively, can be replaced by the term “modification of biomembranelipid composition”, preferably modification of biomembrane lipidcomposition in a eukaryotic host cells, particularly in a fungal hostcell. As mentioned herein, in the context of overexpression of helperproteins, such helper proteins are involved in sphingolipid biosynthesis(helper proteins comprising SEQ ID NOs: 1-9), phospholipid biosynthesis(helper protein comprising SEQ ID NO: 10), lipid transport (helperprotein comprising SEQ ID NO: 11), ergosterol biosynthesis (helperproteins comprising SEQ ID NOs: 12-15), fatty acid biosynthesis,phosphatidic acid biosynthesis and/or phospholipid metabolic process.The term “sphingolipid biosynthesis” as used herein includes fatty acidelongation. Consequently, helper proteins involved in sphingolipidbiosynthesis can be involved in sphingolipid biosynthesis and/or infatty acid elongation. Biosynthesis of complex sphingolipids such asIPC, MIPC or M(IP)₂C is inseparably linked to the elongation of fattyacids. Long chain fatty acids undergo a consecutive 4-step cycle in theprocess of fatty acid elongation leading to the formation of very longchain fatty acids (e.g. C24 or C26). Very long chain fatty acidsproduced by the elongation of fatty acids are then readily incorporatedas direct substrates in the process of sphingolipid biosynthesisyielding complex sphingolipids (IPC, MIPC, M(IP)₂C). The initial stepsin the biosynthesis of sphingolipids occur in the endoplasmic reticulumbut the further modification and maturation process on ceramides toyield complex inositol-containing phosphorylceramides takes place in theGolgi. Therefore, substantial amounts of ceramides and hexosyl-ceramidescan be found in the biomembranes of the endoplasmic reticulum but aswell to a lower extent in further organelles of the secretory route(vesicles and Golgi) and the cellular plasma membrane. Complexsphingolipids (IPC, MIPC, M(IP)₂C) are highest enriched in the cellularplasma membrane co-localizing with sterols in so-called lipid rafts butcan as well be found at lower amounts in the biomembranes of theendoplasmic reticulum and other organelles along the secretory route(vesicles, Golgi). As also mentioned herein, in the context ofunderexpression of helper proteins, such helper proteins are for exampleinvolved in lipid storage (e.g. KO helper proteins comprising SEQ ID NO:16 or 17). Accordingly, modification of biomembrane lipid compositionmay be preferably affected by sphingolipid biosynthesis, phospholipidbiosynthesis, lipid transport, or ergosterol biosynthesis in case ofoverexpression of a helper protein as described herein or may bepreferably affected by lipid storage in case of underexpression of ahelper protein as described herein. Helper proteins of the presentinvention being involved in lipid metabolism are preferably nottranscription factors. The different groups of helper proteins (i.e.involved in sphingolipid biosynthesis, phospholipid biosynthesis, lipidtransport, ergosterol biosynthesis and lipid storage) described hereinare based on Pichia pastoris genes.

The term “protein of interest” as used herein generally relates to anyprotein but preferably relates to a “heterologous protein” or“recombinant protein” and even more preferred to a “non-membrane proteinof interest”. A “non-membrane protein of interest” means that theprotein of interest is preferably not an integral membrane protein whichis in its natural environment (i.e. in situ) permanently part of orattached to biological membranes of an eukaryotic cell, such as fungalcells. Such proteins are classified as spaning across a biologicalmembrane at least once. Accordingly, a non-membrane protein of interestas used herein does preferably not have a transmembrane domain. The term“non-membrane protein” as used herein however includes GPI-anchoredproteins.

As used herein, a “homologue” or “functional homologue” of a helperprotein of the present invention shall mean that a protein has the sameor conserved residues at a corresponding position in their primary,secondary or tertiary structure. The term also extends to two or morenucleotide sequences encoding homologous polypeptides. In particular,polypeptides homologous to the present helper proteins have at leastabout 30% amino acid sequence identity with regard to a sequencedisclosed herein (SEQ ID-Nos 1-18). Preferably, a homologous polypeptidewill have at least about 35% amino acid sequence identity, morepreferably at least about 40% amino acid sequence identity, morepreferably at least about 45% amino acid sequence identity, morepreferably at least about 50% amino acid sequence identity, morepreferably at least about 55% amino acid sequence identity, morepreferably at least about 60% amino acid sequence identity, morepreferably at least about 65% amino acid sequence identity, morepreferably at least about 70% amino acid sequence identity, morepreferably at least about 75% amino acid sequence identity, morepreferably at least about 80% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, morepreferably at least about 90%, such as 91, 92, 93, 94, 95, 96, 97, 98 or99% amino acid sequence identity, more preferably at least about 95%amino acid sequence identity to a native sequence, or any otherspecifically defined fragment of a full-length compound. When thefunction as a helper protein is proven with such a homologue, thehomologue is called “functional homologue”. A functional homologueperforms the same or substantially the same function as the helperprotein from which it is derived from, i.e. it has the same enzymaticactivity or catalyses the same metabolic reaction in the lipidmetabolism of the cell. In the case of nucleotide sequences a“functional homologue” preferably means a nucleotide sequence having asequence different form the original nucleotide sequence, but whichstill codes for the same amino acid sequence, due to the use of thedegenerated genetic code. A functional homologue may also be abiologically active fragment of a helper protein. Generally, abiologically active fragment of a helper protein shall mean a fragmentof said helper protein that exerts a biological effect similar orcomparable to that of the full length helper protein, which means saidbiologically active fragment results in at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90% of the increase of the POIyield observed with the corresponding full length helper protein (SEQID-NOs 1-18). Such active fragment can be obtained e.g. by amino- and/orcarboxy-terminal deletions and/or by deletion of at least 1 amino acidwhich is not the amino- and/or carboxy terminal amino acid of the aminoacid sequence of said full length helper protein.

Generally, homologues can be prepared using any mutagenesis procedureknown in the art, such as site-directed mutagenesis, synthetic geneconstruction, semi-synthetic gene construction, random mutagenesis,shuffling, etc. Site-directed mutagenesis is a technique in which one ormore (e.g., several) mutations are introduced at one or more definedsites in a polynucleotide encoding the parent. Site-directed mutagenesiscan be accomplished in vitro by PCR involving the use of oligonucleotideprimers containing the desired mutation. Site-directed mutagenesis canalso be performed in vitro by cassette mutagenesis involving thecleavage by a restriction enzyme at a site in the plasmid comprising apolynucleotide encoding the parent and subsequent ligation of anoligonucleotide containing the mutation in the polynucleotide. Usuallythe restriction enzyme that digests the plasmid and the oligonucleotideis the same, permitting sticky ends of the plasmid and the insert toligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl.Acad. Sci. USA 76: 4949-4955; and Barton et ai, 1990, Nucleic Acids Res.18: 7349-4966. Site-directed mutagenesis can also be accomplished invivo by methods known in the art. See, e.g., U.S. Patent ApplicationPublication No. 2004/0171 154; Storici et ai, 2001, Nature Biotechnol.19: 773-776; Kren et ai, 1998, Nat. Med. 4: 285-290; and Calissano andMacino, 1996, Fungal Genet. Newslett. 43: 15-16. Synthetic geneconstruction entails in vitro synthesis of a designed polynucleotidemolecule to encode a polypeptide of interest. Gene synthesis can beperformed utilizing a number of techniques, such as the multiplexmicrochip-based technology described by Tian et al. (2004, Nature 432:1050-1054) and similar technologies wherein oligonucleotides aresynthesized and assembled upon photo-programmable microfluidic chips.Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be usedinclude error-prone PCR, phage display (e.g., Lowman et al, 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7:127). Mutagenesis/shuffling methods can be combinedwith high-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides expressed by host cells (Ness et a/.,1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods known in the art. These methodsallow the rapid determination of the importance of individual amino acidresidues in a polypeptide. Semi-synthetic gene construction isaccomplished by combining aspects of synthetic gene construction, and/orsite-directed mutagenesis, and/or random mutagenesis, and/or shuffling.Semisynthetic construction is typified by a process utilizingpolynucleotide fragments that are synthesized, in combination with PCRtechniques. Defined regions of genes may thus be synthesized de novo,while other regions may be amplified using site-specific mutagenicprimers, while yet other regions may be subjected to error-prone PCR ornon-error prone PCR amplification. Polynucleotide subsequences may thenbe shuffled. Alternatively, homologues can be obtained from a naturalsource such as by screening cDNA libraries of other organisms,preferably closely related or related organisms.

The function of a homologue of any one of SEQ ID NOs: 1 to 17 or theadditional helper proteins disclosed herein can be tested by providingexpression cassettes into which the homologue sequences have beeninserted, transforming host cells that carry the sequence encoding atest protein such as one of the model proteins used in the Examplesection or another POI, and determining the difference in the yield ofthe model protein or POI under identical conditions.

The present invention provides an isolated polynucleotide sequenceencoding SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, or 17, or functional homologues of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. The isolated polynucleotidesequence may comprise any one of SEQ ID NOs: 19-35. Preferably, theisolated polynucleotide sequence consists of the nucleotide sequence ofany one of SEQ ID NOs: 30-58. The present invention also provides anisolated polynucleotide sequence encoding PP7435_Chr3-0788 (SUR2),PP7435_Chr3-1005 (PHS1), PP7435_Chr2-0350 (AUR1), PP7435_Chr4-0626(IFA38), PP7435_Chr3-0669 (SCS7), PP7435_Chr3-0636 (CRD1),PP7435_Chr3-0950 (SLC4), PP7435_Chr2-0585 (PIS1), PP7435_chr1-0934(PRY2), ARV1 (ORF not annotated; located between PP7435_Chr4-0493/0494),PP7435_Chr3-0741 (ARE2), PP7435_Chr4-0963 (FAD12), PP7435_Chr1-0794(PSD1), PP7435_Chr1-0160 (SLC1), PP7435_CHR1-0078 (GPT2)PP7435_Chr3-1169 (DGK1) and PP7435_Chr2-0045 (CDS1) or functionalhomologues thereof.

Furthermore, the present invention provides an isolated polypeptidecomprising a polypeptide sequence having at least 30%, such as at least31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, or 18. Furthermore, the present invention provides anisolated polypeptide comprising a polypeptide sequence having at least30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99 or 100% sequence identity to an amino acid sequence selectedfrom the group consisting of PP7435_Chr3-0788 (SUR2), PP7435_Chr3-1005(PHS1), PP7435_Chr2-0350 (AUR1), PP7435_Chr4-0626 (IFA38),PP7435_Chr3-0669 (SCS7), PP7435_Chr3-0636 (CRD1), PP7435_Chr3-0950(SLC4), PP7435_Chr2-0585 (PIS1), PP7435_chr1-0934 (PRY2), ARV1 (ORF notannotated; located between PP7435_Chr4-0493/0494), PP7435_Chr3-0741(ARE2), PP7435_Chr4-0963 (FAD12), PP7435_Chr1-0794 (PSD1),PP7435_Chr1-0160 (SLC1), PP7435_CHR1-0078 (GPT2) PP7435_Chr3-1169 (DGK1)and PP7435_Chr2-0045 (CDS1).

The term “isolated” means a substance in a form or environment that doesnot occur in nature. Non-limiting examples of isolated substancesinclude (1) any non-naturally occurring substance, (2) any substanceincluding, but not limited to, any enzyme, variant, nucleic acid,protein, peptide or cofactor, that is at least partially removed fromone or more or all of the naturally occurring constituents with which itis associated in nature; (3) any substance modified by the hand of manrelative to that substance found in nature, e.g. cDNA made from mRNA; or(4) any substance modified by increasing the amount of the substancerelative to other components with which it is naturally associated(e.g., recombinant production in a host cell; multiple copies of a geneencoding the substance; and use of a stronger promoter than the promoternaturally associated with the gene encoding the substance).

The present invention provides use of any one of the above mentionedisolated polynucleotides for integration in a host cell. Alternatively,if the polynucleotide(s) already exist in the host cell, the host cellcan be manipulated in a way such that they are overexpressed, as will bedescribed later. In another aspect, the invention relates to the use ofsaid polynucleotide for increasing a POI yield from a host cell, whereinthe nucleotide sequence encoding the POI is co-expressed with saidpolynucleotides.

“Sequence identity” or “% identity” refers to the percentage of residuematches between at least two polypeptide or polynucleotide sequencesaligned using a standardized algorithm. Such an algorithm may insert, ina standardized and reproducible way, gaps in the sequences beingcompared in order to optimize alignment between two sequences, andtherefore achieve a more meaningful comparison of the two sequences. Forpurposes of the present invention, the sequence identity between twoamino acid sequences or nucleotide sequences is determined using theNCBI BLAST program version 2.2.29 (Jan. 6, 2014) (Altschul et al.,Nucleic Acids Res. (1997) 25:3389-3402). Sequence identity of two aminoacid sequences can be determined with blastp set at the followingparameters: Matrix: BLOSUM62, Word Size: 3; Expect value: 10; Gap cost:Existence=11, Extension=1; Filter=low complexity activated; FilterString: L; Compositional adjustments: Conditional compositional scorematrix adjustment. For purposes of the present invention, the sequenceidentity between two nucleotide sequences is determined using the NCBIBLAST program version 2.2.29 (Jan. 6, 2014) with blastn set at thefollowing exemplary parameters: Word Size: 11; Expect value: 10; Gapcosts: Existence=5, Extension=2; Filter=low complexity activated;Match/Mismatch Scores: 2,-3; Filter String: L; m.

Moreover, the present invention provides for a host cell engineered tooverexpress a polynucleotide encoding a helper protein of the presentinvention. The helper proteins include any one of SEQ ID NOs: 1-15,respectively, or functional homologues thereof. The helper proteins alsoinclude PP7435_Chr3-0788 (SUR2), PP7435_Chr3-1005 (PHS1),PP7435_Chr2-0350 (AUR1), PP7435_Chr4-0626 (IFA38), PP7435_Chr3-0669(SCS7), PP7435_Chr3-0636 (CRD1), PP7435_Chr3-0950 (SLC4),PP7435_Chr2-0585 (PIS1), PP7435_chr1-0934 (PRY2), ARV1 (ORF notannotated; located between PP7435_Chr4-0493/0494), PP7435_Chr3-0741(ARE2), PP7435_Chr4-0963 (FAD12), PP7435_Chr1-0794 (PSD1),PP7435_Chr1-0160 (SLC1), PP7435_CHR1-0078 (GPT2) PP7435_Chr3-1169 (DGK1)and PP7435_Chr2-0045 (CDS1) or functional homologues thereof. Similarly,the present invention provides for a host cell engineered tounderexpress a polynucleotide encoding a helper protein of the presentinvention (KO helper protein). KO helper proteins include any one of SEQID NOs: 16 or 17, respectively, or functional homologues thereof. A hostcell engineered to underexpress a polynucleotide encoding a KO helperprotein or a helper protein of the present invention may furtheroverexpress one or more helper proteins as set forth in the itemsdisclosed herein.

Preferably, the invention provides a recombinant host cell formanufacturing a protein of interest, wherein the host cell is engineeredto overexpress a polynucleotide encoding a helper protein comprising anamino acid having at least 30% sequence identity to an amino acidsequence as shown in any one of SEQ ID NOs: 1-15.

Preferably, the invention provides a recombinant host cell formanufacturing a protein of interest, wherein the host cell is engineeredto underexpress a polynucleotide encoding a KO helper protein comprisingan amino acid having at least 30% sequence identity to an amino acidsequence as shown in any one of SEQ ID NOs: 16 or 17.

The term “expressing a polynucleotide” means when a polynucleotide istranscribed to mRNA and the mRNA is translated to a polypeptide. Theterm “overexpress” generally refers to any amount greater than or equalto an expression level exhibited by a reference standard. The terms“overexpress,” “overexpressing,” “overexpressed” and “overexpression” inthe present invention refer an expression of a gene product or apolypeptide at a level greater than the expression of the same geneproduct or polypeptide prior to a genetic alteration of the host cell orin a comparable host which has not been genetically altered at definedconditions. In the present invention, a helper protein comprising anamino acid sequence as shown in any one of SEQ ID NOs: 1-15 isoverexpressed. If a host cell does not comprise a given gene product, itis possible to introduce the gene product into the host cell forexpression; in this case, any detectable expression is encompassed bythe term “overexpression.”

The term “underexpress” generally refers to any amount less than anexpression level exhibited by a reference standard, which is the hostcell prior to the engineering to underexpress the KO helper protein asdescribed herein. The terms “underexpress,” “underexpressing,”“underexpressed” and “underexpression” in the present invention refer anexpression of a gene product or a polypeptide at a level less than theexpression of the same gene product or polypeptide prior to a geneticalteration of the host cell or in a comparable host which has not beengenetically altered. For example the KO protein maybe underexpressedcompared to the reference standard by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99% or may be even not expressed at all(reduced by 100%). No expression of the gene product or a polypeptide,for example obtained by deletion/knock-out of the KO helper gene, isalso encompassed by the term “underexpression”.

As used herein, “engineered” host cells are host cells which have beenmanipulated using genetic engineering, i.e. by human intervention. Whena host cell is “engineered to overexpress” a given protein, the hostcell is manipulated such that the host cell has the capability toexpress, preferably overexpress a helper protein or functional homologuethereof, thereby expression of a given protein, e.g. POI or modelprotein is increased compared to the host cell under the same conditionprior to manipulation.

When a host cell “underexpresses” or is “engineered to underexpress” agiven protein, the host cell is manipulated such that the host cell hasthe capability to underexpress a KO helper protein or functionalhomologue thereof, thereby expression of a given protein, e.g. POI ormodel protein is increased compared to the host cell under the samecondition prior to manipulation. Underexpression can be carried out byany method that prevents the functional expression of a KO helperprotein comprising the amino acid sequence as shown in any one of SEQ IDNOs: 16 or 17 or functional homologues thereof. This results in theincapability to exert its function. Means of underexpression may includegene silencing (e.g. RNAi genes antisense), knocking-out, alteringexpression level, altering expression pattern, by mutagenizing the genesequence, disrupting the sequence, insertions, additions, mutations,modifying expression control sequences, and the like. Preferred means ofunderexpression are knocking-out the the functional expression of the KOhelper protein, e.g. by deleting the coding sequence of the KO helperprotein or fragments thereof or by deleting a regulatory sequencerequired for expression of the KO helper protein (e.g. the promoter).

“Prior to engineering” when used in the context of host cells of thepresent invention means that such host cells are not engineered using apolynucleotide encoding a helper protein or functional homologuethereof. Said term thus also means that host cells do not over- orunderexpress a polynucleotide encoding a helper protein or functionalhomologue thereof or are not engineered to over- or underexpress apolynucleotide encoding a helper protein or functional homologuethereof.

The term “engineering said host cell to comprise a heterologouspolynucleotide encoding said protein of interest” as used herein meansthat a host cell of the present invention is equipped with aheterologous polynucleotide encoding a protein of interest, i.e., a hostcell of the present invention is engineered to contain a heterologouspolynucleotide encoding a protein of interest. This can be achieved,e.g., by transformation or transfection or any other suitable techniqueknown in the art for the introduction of a polynucleotide into a hostcell.

Overexpression

Overexpression can be achieved in any ways known to a skilled person inthe art as will be described later in detail. In general, it can beachieved by increasing transcription/translation of the gene, e.g. byincreasing the copy number of the gene or altering or modifyingregulatory sequences or sites associated with expression of a gene, e.g.an endogenous polynucleotide encoding a protein which is involved inlipid metabolism or functional homolog thereof. For example,overexpression can be achieved by introducing one or more copies of thepolynucleotide encoding a helper protein or a functional homologueoperably linked to regulatory sequences (e.g. a promoter). For example,the gene can be operably linked to a strong constitutive promoter and/orstrong ubiquitous promoter in order to reach high expression levels.Such promoters can be endogenous promoters or recombinant promoters.Alternatively, it is possible to remove regulatory sequences such thatexpression becomes constitutive. One can substitute the native promoterof a given gene with a heterologous promoter which increases expressionof the gene or leads to constitutive expression of the gene. Forexample, the helper protein maybe overexpressed by more than 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more than 300% by thehost cell compared to the host cell prior to engineering and culturedunder the same conditions. Using inducible promoters additionally makesit possible to increase the expression in the course of host cellcultivation. Furthermore, overexpression can also be achieved by, forexample, modifying the chromosomal location of a particular gene,altering nucleic acid sequences adjacent to a particular gene such as aribosome binding site or transcription terminator, modifying proteins(e.g., regulatory proteins, suppressors, enhancers, transcriptionalactivators and the like) involved in transcription of the gene and/ortranslation of the gene product, or any other conventional means ofderegulating expression of a particular gene routine in the artincluding but not limited to use of antisense nucleic acid molecules,for example, to block expression of repressor proteins or deleting ormutating the gene for a transcriptional factor which normally repressesexpression of the gene desired to be overexpressed. Prolonging the lifeof the mRNA may also improve the level of expression. For example,certain terminator regions may be used to extend the half-lives of mRNA(Yamanishi et al., Biosci. Biotechnol. Biochem. (2011) 75:2234 and US2013/0244243). If multiple copies of genes are included, the genes caneither be located in plasmids of variable copy number or integrated andamplified in the chromosome. If the host cell does not comprise the geneencoding the helper protein, it is possible to introduce the gene intothe host cell for expression. In this case, “overexpression” meansexpressing the gene product using any methods known to a skilled personin the art.

Those skilled in the art will find relevant instructions in Martin etal. (Bio/Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41(1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)),Eikmanns et al. (Gene 102, 93-98 (1991)), EP 0 472 869, U.S. Pat. No.4,601,893, Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991)),Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)),WO 96/15246, Malumbres et al. (Gene 134, 15-24 (1993)), JP-A-10-229891,Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998))and Makrides (Microbiological Reviews 60, 512-538 (1996)), inter alia,and in well-known textbooks on genetics and molecular biology.

Helper Proteins

The helper proteins of the present invention were originally isolatedfrom Pichia pastoris CBS7435 strain or in case of SEQ ID NO: 8 and 9from Saccharomyces cerevisiae. The methylotrophic yeast Pichia pastoris(Komagataella phaffii) CBS7435 is the parental strain of commonly usedP. pastoris recombinant protein production hosts. Its complete genomicsequence is described in Sturmberger et al. [J. Biotechnol. (2016).235(4):121-131)]. The genes encoding the helper proteins identifiedherein have so far not been associated with a beneficial effect onprotein yield. The complete genomic sequence of very closely relatedPichia pastoris strain, GS115, is almost identical to the genomicsequence of CBS7435 (Nat. Biotechnol. 27 (6), 561-566) and the sequencesof GS115 corresponding to the sequences of CBS7435 might also be used inthe invention.

It is envisioned that the helper proteins can be overexpressed over awide range of host cells. Thus, instead of using the sequences native tothe species or the genus, the helper protein sequences may also be takenor derived from other prokaryotic or eukaryotic organisms. The foreignDNA sequences encoding the helper proteins may be obtained from avariety of sources, such as from a plant, insect, fungal or mammalianspecies, preferably from the class of Saccharomycetes, preferably fromthe order of Saccharomycetales, preferably from the family ofSaccharomycetaceae, and preferably from the genus of Komagataella.

In particular, the invention refers to a genetically modified host cellwhich is capable of overexpressing the helper proteins comprising anamino acid sequence as shown in any one of SEQ ID NOs: 1-15 orfunctional homologues thereof alone or combinations thereof. Host cellsbeing engineered to reflect a combination of helper proteins areenvisaged in a preferred embodiment. These host cells are preferablyapplied in the methods and uses described herein. A combination includesthat 2 or more, such as 2, 3, 4, 5, 6, 7, 8 or more, helper proteins ora functional homologue thereof are chosen from any one of SEQ ID NOs:1-15.

Likewise, a combination includes a combination of one or more helperprotein(s) chosen from any one of SEQ ID NOs: 1-15 and PP7435_Chr3-0788(SUR2), PP7435_Chr3-1005 (PHS1), PP7435_Chr2-0350 (AUR1),PP7435_Chr4-0626 (IFA38), PP7435_Chr3-0669 (SCS7), PP7435_Chr3-0636(CRD1), PP7435_Chr3-0950 (SLC4), PP7435_Chr2-0585 (PIS1),PP7435_chr1-0934 (PRY2), ARV1 (ORF not annotated; located betweenPP7435_Chr4-0493/0494), PP7435_Chr3-0741 (ARE2), PP7435_Chr4-0963(FAD12), PP7435_Chr1-0794 (PSD1), PP7435_Chr1-0160 (SLC1),PP7435_CHR1-0078 (GPT2) PP7435_Chr3-1169 (DGK1) and PP7435_Chr2-0045(CDS1) or a functional homologue thereof, which are overexpressed, and ahelper protein chosen from any one of SEQ ID NOs: 16 or 17 or afunctional homologue thereof, which are underexpressed. Host cells beingengineered to reflect such a combination are envisaged in a preferredembodiment. These host cells may be applied in the methods and usesdescribed herein.

The following helper proteins and combinations of helper proteins of thepresent invention involved in sphingolipid biosynthesis are preferablyapplied in host cell, methods and uses described herein:

-   (a) protein comprising the amino acid sequence as shown in SEQ ID    NO: 1 or a functional homologue thereof, wherein the functional    homologue has at least 30% sequence identity to an amino acid    sequence as shown in SEQ ID NO: 1;-   (b) protein comprising the amino acid sequence as shown in SEQ ID    NO: 2 or a functional homologue thereof, wherein the functional    homologue has at least 30% sequence identity to an amino acid    sequence as shown in SEQ ID NO: 2;-   (c) protein comprising the amino acid sequence as shown in SEQ ID    NO: 3 or a functional homologue thereof, wherein the functional    homologue has at least 30% sequence identity to an amino acid    sequence as shown in SEQ ID NO: 3;-   (d) proteins comprising the amino acid sequence as shown in SEQ ID    NOs: 1 and 2 or a functional homologue thereof, wherein the    functional homologue has at least 30% sequence identity to an amino    acid sequence as shown in SEQ ID NOs: 1 and 2, respectively;-   (e) proteins comprising the amino acid sequence as shown in SEQ ID    NOs: 1 and 3 or a functional homologue thereof, wherein the    functional homologue has at least 30% sequence identity to an amino    acid sequence as shown in SEQ ID NOs: 1 and 3, respectively;-   (f) proteins comprising the amino acid sequence as shown in SEQ ID    NOs: 1, 3 and 4 or a functional homologue thereof, wherein the    functional homologue has at least 30% sequence identity to an amino    acid sequence as shown in SEQ ID NOs: 1, 3 and 4, respectively;-   (g) proteins comprising the amino acid sequence as shown in SEQ ID    NOs: 1, 3, 4 and 8 or a functional homologue thereof, wherein the    functional homologue has at least 30% sequence identity to an amino    acid sequence as shown in SEQ ID NOs: 1, 3, 4 and 8, respectively;-   (h) proteins comprising the amino acid sequence as shown in SEQ ID    NOs: 5 and 6 or a functional homologue thereof, wherein the    functional homologue has at least 30% sequence identity to an amino    acid sequence as shown in SEQ ID NOs: 5 and 6, respectively;-   (i) proteins comprising the amino acid sequence as shown in SEQ ID    NOs: 1 and 7 or a functional homologue thereof, wherein the    functional homologue has at least 30% sequence identity to an amino    acid sequence as shown in SEQ ID NOs: 1 and 7, respectively;-   (j) proteins comprising the amino acid sequence as shown in SEQ ID    NOs: 5, 6 and 9 or a functional homologue thereof, wherein the    functional homologue has at least 30% sequence identity to an amino    acid sequence as shown in SEQ ID NOs: 5, 6 and 9, respectively;-   (k) proteins comprising the amino acid sequence as shown in SEQ ID    NOs: 1, 5, 6 and 9 or a functional homologue thereof, wherein the    functional homologue has at least 30% sequence identity to an amino    acid sequence as shown in SEQ ID NOs: 1, 5, 6 and 9, respectively.

A combination of helper proteins of the present invention includesoverexpressing at least one protein involved in sphingolipidbiosynthesis and at least one protein involved in lipid transport. In apreferred combination applied in host cell, methods and uses describedherein, the protein involved in sphingolipid biosynthesis comprises anamino acid sequence as shown in SEQ ID NO: 1 or a functional homologuethereof and the protein involved in lipid transport comprises an aminoacid sequence as shown in SEQ ID NO: 11 or a functional homologuethereof, wherein the functional homologue has at least 30% sequenceidentity to an amino acid sequence as shown in SEQ ID NOs: 1 and 11,respectively.

A combination of helper proteins of the present invention includesoverexpressing at least one protein involved in phospholipidbiosynthesis and at least one protein involved in lipid transport. In apreferred combination applied in host cell, methods and uses describedherein, the protein involved in phospholipid biosynthesis comprises anamino acid sequence as shown in SEQ ID NO: 10 or a functional homologuethereof and the protein involved in lipid transport comprises an aminoacid sequence as shown in SEQ ID NO: 11 or a functional homologuethereof, wherein the functional homologue has at least 30% sequenceidentity to an amino acid sequence as shown in SEQ ID NOs: 10 and 11,respectively.

The present invention further pertains to overexpressing a chaperone inaddition to the helper proteins of the present invention. The term“chaperone as used herein relates to polypeptides that assist thefolding, unfolding, assembly or disassembly of other polypeptides.Preferably, the chaperone comprises an amino acid sequence as shown inSEQ ID NO: 18 or a functional homologue thereof, wherein the functionalhomologue has at least 30% sequence identity to an amino acid sequenceas shown in SEQ ID NO: 18.

Accordingly, a combination of helper proteins of the present inventionincludes overexpressing at least one protein involved in sphingolipidbiosynthesis and at least one chaperone. Preferably, the proteininvolved in sphingolipid biosynthesis comprises an amino acid sequenceas shown in SEQ ID NO: 1 or a functional homologue thereof and thechaperone comprises an amino acid sequence as shown in SEQ ID NO: 18 ora functional homologue thereof, wherein the functional homologue has atleast 30% sequence identity to an amino acid sequence as shown in SEQ IDNOs: 1 and 18, respectively.

A further combination of helper proteins of the present inventionincludes overexpressing at least one protein involved in ergosterolbiosynthesis and at least one chaperone. Preferably, the proteininvolved in ergosterol biosynthesis comprises an amino acid sequence asshown in SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 or a functionalhomologue thereof and the chaperone comprises an amino acid sequence asshown in SEQ ID NO: 18 or a functional homologue thereof, wherein thefunctional homologue has at least 30% sequence identity to an amino acidsequence as shown in SEQ ID NOs: 13 and 18, respectively.

Protein of Interest

The term “protein of interest” (POI) as used herein refers to a proteinthat is produced by means of recombinant technology in a host cell. Morespecifically, the protein may either be a polypeptide not naturallyoccurring in the host cell, i.e. a heterologous protein, or else may benative to the host cell, i.e. a homologous protein to the host cell, butis produced, for example, by transformation with a self-replicatingvector containing the nucleic acid sequence encoding the POI, or uponintegration by recombinant techniques of one or more copies of thenucleic acid sequence encoding the POI into the genome of the host cell,or by recombinant modification of one or more regulatory sequencescontrolling the expression of the gene encoding the POI, e.g. of thepromoter sequence. In general, the proteins of interest referred toherein may be produced by methods of recombinant expression well knownto a person skilled in the art. A protein of interest is preferably nota membrane protein, i.e. a POI preferably is a non-membrane protein ofinterest.

Host Cell

As used herein, a “host cell” refers to a cell which is capable ofprotein expression and optionally protein secretion. Such host cell isapplied in the methods of the present invention. For that purpose, forthe host cell to over- or underexpress a polypeptide which is involvedin lipid metabolism, a nucleotide sequence encoding said polypeptide ispresent or introduced in the cell. Host cells provided by the presentinvention can be eukaryotic host cells. More preferred are non-mammalianeukaryotic host cells. Even more preferred are fungal host cells. Aswill be appreciated by one of skill in the art, a prokaryotic cell lacksa membrane-bound nucleus, while a eukaryotic cell has a membrane-boundnucleus. Examples of eukaryotic cells include, but are not limited to,vertebrate cells, mammalian cells, human cells, animal cells,invertebrate cells, plant cells, nematodal cells, insect cells, stemcells, fungal cells or yeast cells.

Examples of yeast cells include but are not limited to the Saccharomycesgenus (e.g. Saccharomyces cerevisiae, Saccharomyces kluyveri,Saccharomyces uvarum), the Komagataella genus (Komagataella pastoris,Komagataella pseudopastoris or Komagataella phaffii), Kluyveromycesgenus (e.g. Kluyveromyces lactis, Kluyveromyces marxianus), the Candidagenus (e.g. Candida utilis, Candida cacaos), the Geotrichum genus (e.g.Geotrichum fermentans), as well as Hansenula polymorpha and Yarrowialipolytica,

The genus Pichia is of particular interest. Pichia comprises a number ofspecies, including the species Pichia pastoris, Pichia methanolica,Pichia kluyveri, and Pichia angusta. Most preferred is the speciesPichia pastoris.

The former species Pichia pastoris has been divided and renamed toKomagataella pastoris and Komagataella phaffii. Therefore Pichiapastoris is synonymous for both Komagataella pastoris and Komagataellaphaffii.

Examples for Pichia pastoris strains useful in the present invention areX33 and its subtypes GS115, KM71, KM71H; CBS7435 (mut+) and its subtypesCBS7435 mut^(S), CBS7435 mut^(S)ΔArg, CBS7435 mut^(S)ΔHis, CBS7435mut^(S)ΔArg, ΔHis, CBS7435 mut^(S) PDI⁺, CBS704 (=NRRL Y-1603=DSMZ70382), CBS 2612 (=NRRL Y-7556), CBS 9173-9189 and DSMZ 70877 as well asmutants thereof. These yeast strains are available from cellrepositories such as the American Tissue Culture Collection (ATCC), the“Deutsche Sammlung von Mikroorganismen und Zellkulturen” (DSMZ) inBraunschweig, Germany, or from the Dutch “Centraalbureau voorSchimmelcultures” (CBS) in Uetrecht, The Netherlands.

According a further preferred embodiment, the host cell is a Pichiapastoris, Hansenula polymorpha, Trichoderma reesei, Saccharomycescerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichiamethanolica, Candida boidinii, and Komagataella, and Schizosaccharomycespombe. It may also be a host cell from Ustilago maydis. These yeaststrains are available from cell repositories such as the American TissueCulture Collection (ATCC), the “Deutsche Sammlung von Mikroorganismenund Zellkulturen” (DSMZ) in Braunschweig, Germany, or from the Dutch“Centraalbureau voor Schimmelcultures” (CBS) in Uetrecht, TheNetherlands.

Examples of E. coli include those derived from Escherichia coli K12strain, specifically, HMS 174, HMS174 (DE3), NM533, XL1-Blue, C600, DH1,HB101, JM109, as well as those derived from B-strains, specificallyBL-21, BL21 (DE3) and the like. E. coli cells may be used, for example,for cloning purposes, but may also be used as host cells of the presentinvention. These bacterial strains are available from cell repositoriessuch as the American Tissue Culture Collection (ATCC), the “DeutscheSammlung von Mikroorganismen und Zellkulturen” (DSMZ) in Braunschweig,Germany, or from the Dutch “Centraalbureau voor Schimmelcultures” (CBS)in Uetrecht, The Netherlands.

Preferably, the helper proteins expressed by the host cell is from thesame cell or recombined from a cell of the same species, genus orfamily. As used herein, “recombinant” refers to the alteration ofgenetic material by human intervention. Typically, recombinant refers tothe manipulation of DNA or RNA in a virus, cell, plasmid or vector bymolecular biology (recombinant DNA technology) methods, includingcloning and recombination. A recombinant cell, polypeptide, or nucleicacid can be typically described with reference to how it differs from anaturally occurring counterpart (the “wild-type”). A “recombinant cell”or “recombinant host cell” refers to a cell or host cell that has beengenetically altered to comprise a nucleic acid sequence which was notnative to said cell.

The term “manufacture” or “manufacturing” as used presently refers tothe process in which the protein of interest is expressed. A “host cellfor manufacturing a protein of interest” refers to a host cell in whichnucleic acid sequences encoding a protein of interest may be introduced.The recombinant host cell within the present invention does notnecessarily contain the nucleic acid sequences encoding a protein ofinterest. It is appreciated by a skilled person in the art that the hostcells can be provided for inserting desired nucleotide sequences intothe host cell, for example, in a kit. “Manufacturing” or “manufacture”also refers to a process of producing a POI using a host cell of thepresent invention, cultivating the host cell of the present inventionunder suitable conditions to overexpress the protein involved in lipidmetabolism or functional homologue thereof and express said protein ofinterest, and optionally isolating said protein of interest from thecell culture.

The term “nucleotide sequence” or “nucleic acid sequence” used hereinrefers to either DNA or RNA. “Nucleic acid sequence” or “polynucleotidesequence” or simply “polynucleotide” refers to a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesread from the 5′ to the 3′ end. It includes both self-replicatingplasmids, infectious polymers of DNA or RNA, and non-functional DNA orRNA.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

The terms “polypeptide” and “protein” are interchangeably used. The term“polypeptide” refers to a protein or peptide that contains two or moreamino acids, typically at least 3, preferably at least 20, morepreferred at least 30, such as at least 50 amino acids. Accordingly, apolypeptide comprises an amino acid sequence, and, thus, sometimes apolypeptide comprising an amino acid sequence is referred to herein as a“polypeptide comprising a polypeptide sequence”. Thus, herein the term“polypeptide sequence” is interchangeably used with the term “amino acidsequence”. As mentioned, overexpression can be achieved by insertion ofone or more than one extra copy of the selected helper protein.According to a preferred embodiment, the polynucleotide encoding thehelper protein can be presented in a single copy or in multiple copiesper cell. The copies may be adjacent to or distant from each other.According to another preferred embodiment, the method of the inventionemploys recombinant nucleotide sequences encoding the helper proteinsprovided on one or more plasmids suitable for integration into thegenome of the host cell, in a single copy or in multiple copies percell. The copies may be adjacent to or distant from each other.Overexpression can be in one embodiment achieved by expressing one ormultiple copies of the polynucleotide, such as 2, 3, 4, 5, 6 or morecopies of said polynucleotide per host cell. The polynucleotides arepreferably operably linked to transcriptional and translationalregulatory sequences that provide for expression of the polypeptide inthe host cells. The term “transcriptional regulatory sequences” as usedherein refers to nucleotide sequences that are associated with a genenucleic acid sequence and which regulate the transcription of the gene.The term “translational regulatory sequences” as used herein refers tonucleotide sequences that are associated with a gene nucleic acidsequence and which regulate the translation of the gene. Transcriptionaland/or translational regulatory sequences can either be located inplasmids or vectors or integrated in the chromosome of the host cell.Transcriptional and/or translational regulatory sequences are located inthe same nucleic acid molecule of the gene which it regulates.Preferably, the overexpression can be achieved by having 1, 2, 3, 4 ormore copies of a polynucleotide encoding a helper protein comprising anamino acid sequence shown in any one of SEQ ID NOs: 1-15 or functionalhomologues thereof per host cell or by having 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 copies of a polynucleotideencoding a helper protein comprising an amino acid sequence shown in anyone of SEQ ID NOs: 1-15 or functional homologues thereof per host cell.

The polynucleotide encoding the helper protein and/or the polynucleotideencoding the POI is/are preferably integrated into the genome of thehost cell. The term “genome” generally refers to the whole hereditaryinformation of an organism that is encoded in the DNA (or RNA forcertain viral species). It may be present in the chromosome, on aplasmid or vector, or both. Preferably, the polynucleotide encoding thehelper protein is integrated into the chromosome of said cell.

The polynucleotide encoding the helper protein or functional homologuethereof may be integrated in its natural locus. “Natural locus” meansthe location on a specific chromosome, where the polynucleotide encodingthe helper protein is located, for example at the natural locus of thegene encoding a helper protein of the present invention. Any such genecan be identified in accordance with the gene identifier as shown inTable 1, above. However, in another embodiment, the polynucleotideencoding the helper protein is present in the genome of the host cellnot at their natural locus, but integrated ectopically. The term“ectopic integration” means the insertion of a nucleic acid into thegenome of a microorganism at a site other than its usual chromosomallocus, i.e., predetermined or random integration. In the alternative,the polynucleotide encoding the helper protein or functional homologuethereof may be integrated in its natural locus and ectopically.

For yeast cells, the polynucleotide encoding the helper protein and/orthe polynucleotide encoding the POI may be inserted into a desiredlocus, such as but not limited to AOX1, GAP, ENO1, TEF, HIS4 (Zamir etal., Proc. NatL Acad. Sci. USA (1981) 78(6):3496-3500), HO (Voth et al.Nucleic Acids Res. 2001 Jun. 15; 29(12): e59), TYR1 (Mirisola et al.,Yeast 2007; 24: 761-766), His3, Leu2, Ura3 (Taxis et al., BioTechniques(2006) 40:73-78), Lys2, ADE2, TRP1, GAL1, ADH1 or on the integration of5S ribosomal RNA gene.

In other embodiments, the polynucleotide encoding the helper proteinand/or the polynucleotide encoding the POI can be integrated in aplasmid or vector. The terms “plasmid” and “vector” include autonomouslyreplicating nucleotide sequences as well as genome integratingnucleotide sequences. A skilled person is able to employ suitableplasmids or vectors depending on the host cell used.

Preferably, the plasmid is a eukaryotic expression vector, preferably ayeast expression vector.

Plasmids can be used for the transcription of cloned recombinantnucleotide sequences, i.e. of recombinant genes and the translation oftheir mRNA in a suitable host organism. Plasmids can also be used tointegrate a target polynucleotide into the host cell genome by methodsknown in the art, such as described by J. Sambrook et al., MolecularCloning: A Laboratory Manual (3rd edition), Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, New York (2001). A“plasmid” usually comprise an origin for autonomous replication in thehost cells, selectable markers, a number of restriction enzyme cleavagesites, a suitable promoter sequence and a transcription terminator,which components are operably linked together. The polypeptide codingsequence of interest is operably linked to transcriptional andtranslational regulatory sequences that provide for expression of thepolypeptide in the host cells.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence on the same nucleic acidmolecule. For example, a promoter is operably linked with a codingsequence of a recombinant gene when it is capable of effecting theexpression of that coding sequence.

Most plasmids exist in only one copy per bacterial cell. Some plasmids,however, exist in higher copy numbers. For example, the plasmid ColE1typically exists in 10 to 20 plasmid copies per chromosome in E. coli.If the nucleotide sequences of the present invention are contained in aplasmid, the plasmid may have a copy number of 1-10, 10-20, 20-30,30-100 or more per host cell. With a high copy number of plasmids, it ispossible to overexpress helper proteins by the cell.

Large numbers of suitable plasmids or vectors are known to those ofskill in the art and many are commercially available. Examples ofsuitable vectors are provided in Sambrook et al, eds., MolecularCloning: A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring HarborLaboratory (1989), and Ausubel et al, eds., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., New York (1997).

A vector or plasmid of the present invention encompass yeast artificialchromosome, which refers to a DNA construct that can be geneticallymodified to contain a heterologous DNA sequence (e.g., a DNA sequence aslarge as 3000 kb), that contains telomeric, centromeric, and origin ofreplication (replication origin) sequences.

A vector or plasmid of the present invention also encompasses bacterialartificial chromosome (BAC), which refers to a DNA construct that can begenetically modified to contain a heterologous DNA sequence (e.g., a DNAsequence as large as 300 kb), that contains an origin of replicationsequence (Ori), and may contain one or more helicases (e.g., parA, parB,and parC).

Examples of plasmids using yeast as a host include YIp type vector, YEptype vector, YRp type vector, YCp type vector (Yxp vectors are e.g.described in Romanos et al. 1992, Yeast. 8(6):423-488), pGPD-2(described in Bitter et al., 1984, Gene, 32:263-274), pYES, pAO815,pGAPZ, pGAPZα, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, pPICZ, pPICZα,pPIC3K, pPINK-HC, pPINK-LC (all available from Thermo FisherScientific/Invitrogen), pHWO10 (described in Waterham et al., 1997,Gene, 186:37-44), pPZeoR, pPKanR, pPUZZLE and pPUZZLE-derivatives(described in Stadlmayr et al., 2010, J Biotechnol. 150(4):519-29.; Marxet al. 2009, FEMS Yeast Res. 9(8):1260-70.); pJ-vectors (e.g. pJAN,pJAG, pJAZ and their derivatives; all available from BioGrammatics,Inc), pJexpress-vectors, pD902, pD905, pD915, pD912 and theirderivatives, pD12xx, pJ12xx (all available from ATUM/DNA2.0), pRGplasmids (described in Gnijgge et al., 2016, Yeast 33:83-98) 2 μmplasmids (described e.g. in Ludwig et al., 1993, Gene 132(1):33-40).Such vectors are known and are for example described in Cregg et al.,2000, Mol Biotechnol. 16(1):23-52 or Ahmad et al. 2014., Appl MicrobiolBiotechnol. 98(12):5301-17. Additionally suitable vectors can be readilygenerated by advanced modular cloning techniques as for exampledescribed by Lee et al. 2015, ACS Synth Biol. 4(9):975-986; Agmon et al.2015, ACS Synth. Biol., 4(7):853-859; or Wagner and Alper, 2016, FungalGenet Biol. 89:126-136. Additionally, these and other suitable vectorsmay be also available from Addgene.

Examples of plasmids using Escherichia coli as their host include pBR322(available e.g. from New England Biolabs and ThermoFisher Scientific),pBAD-vectors, pET-vector series (both available from e.g. ThermoFisherScientific, pET vectors also from Novagen), pUC18, pUC19, pUC118/119(all available from e.g Takara), pVC119 (described in Del Sol et al.,2006, J Bacteriol. 188(4):1540-1550), pSP64, pSP65 (both from Promega),pTZ-18R/-18U (from Amersham), pTZ-19R/-19U (available fromSigma-Aldrich), pGEM-3, pGEM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf(−) (pGEMvectors are available from Promega), and pBluescript KS (available fromAgilent). Examples of plasmids suitable for expression in Escherichiacoli include, pKK223 (described in Brosius and Holy, 1984, Proc. Natl.Acad. Sci. U.S.A. 81:6929-6933), pMC1403, pMC931 (both described inCasadaban et al. 1980, J Bacteriol. 143(2):971-980), and pKC30(described in Rao, 1984, Gene 31(1-3):247-250). Additionally, these andother suitable vectors may be also available from Addgene.

Promoter

Overexpression of the endogenous polypeptide in the recombinant cell canbe achieved by modifying transcriptional and translational regulatorysequences, including, for example, promoters, enhancers, polyadenylationsignals, transcription terminators, internal ribosome entry sites(IRES), and the like, that provide for the expression of thepolynucleotide sequence in a host cell. Such sequences interactspecifically with cellular proteins involved in transcription (Maniatiset al., Science, 236: 1237-1245 (1987)). Exemplary sequences aredescribed in, for example, Goeddel, Gene Expression Technology: Methodsin Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

For example, overexpression of the endogenous helper protein in therecombinant cell can be achieved by modifying the promoters, forexample, by replacing the endogenous promoter which is operably linkedto the helper protein with another stronger promoter in order to reachhigh expression levels. Such promoter may be inducible or constitutive.Modification of endogenous promoter may be performed by mutation orhomologous recombination using methods known in the art.

The overexpression of the polynucleotide encoding the helper proteins,can be achieved by other methods known in the art, for example bygenetically modifying their endogenous regulatory regions, as describedby Marx et al., 2008 (Marx, H., Mattanovich, D. and Sauer, M. MicrobCell Fact 7 (2008): 23), and Pan et al., 2011 (Pan et al., FEMS YeastRes. (2011) May; (3):292-8.), such methods include, for example,integration of a recombinant promoter that increases expression of thehelper proteins. Transformation is described in Cregg et al. (1985) Mol.Cell. Biol. 5:3376-3385. A “recombinant” promoter is referred to withrespect to the sequence whose expression it drives. As used herein, arecombinant promoter means when the promoter is not a native promoter tothe given sequence, i.e., when the promoter is different from anaturally occurring promoter (the “native promoter”). Such a promoter issometimes also referred to herein as heterologous promoter.

The term “promoter” as used herein refers to a region that facilitatesthe transcription of a particular gene. A promoter typically increasesthe amount of recombinant product expressed from a nucleotide sequenceas compared to the amount of the expressed recombinant product when nopromoter exists. A promoter from one organism can be utilized to enhancerecombinant product expression from a sequence that originates fromanother organism. The promoter can be integrated into a host cellchromosome by homologous recombination using methods known in the art(e.g. Datsenko et al, Proc. Natl. Acad. Sci. U.S.A., 97(12): 6640-6645(2000)). In addition, one promoter element can increase the amount ofproducts expressed for multiple sequences attached in tandem. Hence, onepromoter element can enhance the expression of one or more recombinantproducts.

Promoter activity may be assessed by its transcriptional efficiency.This may be determined directly by measurement of the amount of mRNAtranscription from the promoter, e.g. by Northern Blotting, quantitativePCR or indirectly by measurement of the amount of gene product expressedfrom the promoter.

The promoter could be an “inducible promoter” or “constitutivepromoter.” “Inducible promoter” refers to a promoter which can beinduced by the presence or absence of certain factors, and “constitutivepromoter” refers to an unregulated promoter that allows for continuoustranscription of its associated gene or genes.

In a preferred embodiment, both the transcription of the nucleotidesequences encoding the helper protein and the POI are each driven by aninducible promoter. In another preferred embodiment, both thetranscription of the nucleotide sequences encoding the helper proteinand the POI are each driven by a constitutive promoter. In yet anotherpreferred embodiment, the transcription of the nucleotide sequenceencoding the helper protein is driven by a constitutive promoter and thetranscription of the nucleotide sequence encoding the POI is driven byan inducible promoter. In yet another preferred embodiment, thetranscription of the nucleotide sequences encoding the helper protein isdriven by an inducible promoter and the transcription of the nucleotidesequence encoding the POI is driven by a constitutive promoter. As anexample, the transcription of the helper protein gene may be driven by aconstitutive GAP promoter and the transcription of the nucleotidesequence encoding the POI may be driven by an inducible AOX1 promoter.In one embodiment, the the transcription of the nucleotide sequencesencoding the helper protein and the POI is driven by the same promoteror similar promoters in terms of promoter activity and/or expressionbehaviour.

Many inducible promoters are known in the art. Many are described in areview by Gatz, Curr. Op. Biotech., 7: 168 (1996) (see also Gatz, Ann.Rev. Plant. Physiol. Plant Mol. Biol., 48:89 (1997)). Examples includetetracycline repressor system, Lac repressor system, copper-induciblesystems, salicylate-inducible systems (such as the PR1 a system),glucocorticoid-inducible (Aoyama et al., 1997), alcohol-induciblesystems, e.g., AOX promoters, and ecdysone-inducible systems. Alsoincluded are the benzene sulphonamide-inducible (U.S. Pat. No.5,364,780) and alcohol-inducible (WO 97/06269 and WO 97/06268) induciblesystems and glutathione S-transferase promoters.

Suitable promoter sequences for use with yeast host cells are describedin Mattanovich et al., Methods Mol. Biol. (2012) 824:329-58 and includeglycolytic enzymes like triosephosphate isomerase (TPI),phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase(GAPDH or GAP) and variants thereof, lactase (LAC) and galactosidase(GAL), P. pastoris glucose-6-phosphate isomerase promoter (PPGI), the3-phosphoglycerate kinase promoter (PPGK), the glycerol aldehydephosphate dehydrogenase promoter (PGAP), translation elongation factorpromoter (PTEF), and the promoters of P. pastoris enolase 1 (PENO1),triose phosphate isomerase (PTPI), ribosomal subunit proteins (PRPS2,PRPS7, PRPS31, PRPL1), alcohol oxidase promoter (PAOX) or variantsthereof with modified characteristics, the formaldehyde dehydrogenasepromoter (PFLD), isocitrate lyase promoter (PICL), alpha-ketoisocaproatedecarboxylase promoter (PTHI), the promoters of heat shock proteinfamily members (PSSA1, PHSP90, PKAR2), 6-Phosphogluconate dehydrogenase(PGND1), phosphoglycerate mutase (PGPM1), transketolase (PTKL1),phosphatidylinositol synthase (PPIS1), ferro-O2-oxidoreductase (PFET3),high affinity iron permease (PFTR1), repressible alkaline phosphatase(PPHO8), N-myristoyl transferase (PNMT1), pheromone responsetranscription factor (PMCM1), ubiquitin (PUB14), single-stranded DNAendonuclease (PRAD2), the promoter of the major ADP/ATP carrier of themitochondrial inner membrane (PPET9) (WO2008/128701) and the formatedehydrogenase (FMD) promoter. The GAP promoter, AOX promoter or apromoter derived from GAP or AOX promoter is particularly preferred. AOXpromoters can be induced by methanol and are repressed by e.g. glucose.

Further examples of suitable promoters include Saccharomyces cerevisiaeenolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1),Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase (PGK), and the maltase gene promoter (MAL).

Other useful promoters for yeast host cells are described by Romanos etal, 1992, Yeast 8:423-488.

Suitable promoter sequences for use with E. coli include T7 promoter, T5promoter, tryptophan (trp) promoter, lactose (lac) promoter,tryptophan/lactose (tac) promoter, lipoprotein (Ipp) promoter, and λphage PL promoter in plasmids.

The promoter which drives the expression of the polynucleotide encodingthe helper protein is preferably not the endogenous to the promoter ofthe helper gene. Preferably, a recombinant promoter is used instead ofthe endogenous promoter of the helper protein gene.

Enhancer

In a preferred embodiment, the overexpression is achieved by using anenhancer to enhance the promoter activity which drives the expression ofthe helper protein. Transcriptional enhancers are relatively orientationand position independent, having been found 5′ and 3′ to thetranscription unit, within an intron, as well as within the codingsequence itself. The enhancer may be spliced into the expression vectorat a position 5′ or 3′ to the coding sequence, but is preferably locatedat a site 5′ from the promoter. Most yeast genes contain only one UAS,which generally lies within a few hundred base pairs of the cap site andmost yeast enhancers (UASs) cannot function when located 3′ of thepromoter, but enhancers in higher eukaryotes can function both 5′ and 3′of the promoter.

Many enhancer sequences are now known from mammalian genes (globin, RSV,SV40, EMC, elastase, albumin, a-fetoprotein and insulin). One may alsouse an enhancer from a eukaryotic cell virus, such as the SV40 lateenhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. Yeast enhancers, also called upstream activating sequences(UASs), such as the UAS/GAL system from Saccharomyces cerevisiae, can beadvantageously used with yeast promoters (described in European PatentNo. 0317254 and Rudoni et al., The International Journal of Biochemistryand Cell Biology, (2000), 32(2):215-224).

In a preferred embodiment, 2, 3, 4, 5, 6, 7, 8, 9 or more types ofhelper proteins disclosed by present invention are overexpressed. Forexample, the host cell can be engineered to overexpress 2, 3, 4, 5, 6,7, 8, 9 or more of helper proteins selected from any one of SEQ ID NOs:1-15 or functional homologues thereof, where a functional homologuethereof comprises an amino acid having at least 30% sequence identity toany one of SEQ ID NOs: 1-15, respectively. In a further embodiment thehost cell can be engineered to overexpress 2, 3, 4, 5, 6, 7, 8, 9 ormore of helper proteins selected from any one of SEQ ID NOs: 1-15,PP7435_Chr3-0788 (SUR2), PP7435_Chr3-1005 (PHS1), PP7435_Chr2-0350(AUR1), PP7435_Chr4-0626 (IFA38), PP7435_Chr3-0669 (SCS7),PP7435_Chr3-0636 (CRD1), PP7435_Chr3-0950 (SLC4), PP7435_Chr2-0585(PIS1), PP7435_chr1-0934 (PRY2), ARV1 (ORF not annotated; locatedbetween PP7435_Chr4-0493/0494), PP7435_Chr3-0741 (ARE2),PP7435_Chr4-0963 (FAD12), PP7435_Chr1-0794 (PSD1), PP7435_Chr1-0160(SLC1), PP7435_CHR1-0078 (GPT2) PP7435_Chr3-1169 (DGK1) andPP7435_Chr2-0045 (CDS1) or functional homologues thereof, where afunctional homologue thereof comprises an amino acid having at least 30%sequence identity to any one of SEQ ID NOs: 1-15, PP7435_Chr3-0788(SUR2), PP7435_Chr3-1005 (PHS1), PP7435_Chr2-0350 (AUR1),PP7435_Chr4-0626 (IFA38), PP7435_Chr3-0669 (SCS7), PP7435_Chr3-0636(CRD1), PP7435_Chr3-0950 (SLC4), PP7435_Chr2-0585 (PIS1),PP7435_chr1-0934 (PRY2), ARV1 (ORF not annotated; located betweenPP7435_Chr4-0493/0494), PP7435_Chr3-0741 (ARE2), PP7435_Chr4-0963(FAD12), PP7435_Chr1-0794 (PSD1), PP7435_Chr1-0160 (SLC1),PP7435_CHR1-0078 (GPT2) PP7435_Chr3-1169 (DGK1) and PP7435_Chr2-0045(CDS1), respectively.

Protein of Interest

It is envisioned that when the host cell may be cultured under asuitable condition for the overexpression of the helper protein and theprotein of interest, the host cell would express the protein of interestand overexpresses the polynucleotide encoding a helper proteincomprising an amino acid sequence as shown in any one of SEQ ID NOs:1-15 or a functional homologue thereof, wherein the functional homologuehas at least 30% sequence identity to an amino acid sequence as shown inany one of SEQ ID NOs: 1-15, respectively. It is further envisioned thatwhen the host cell may be cultured under a suitable condition for thecoexpression of the helper protein and the protein of interest, the hostcell would express the protein of interest and overexpresses thepolynucleotide encoding a helper protein comprising an amino acidsequence as shown in any one of SEQ ID NOs: 1-15, PP7435_Chr3-0788(SUR2), PP7435_Chr3-1005 (PHS1), PP7435_Chr2-0350 (AUR1),PP7435_Chr4-0626 (IFA38), PP7435_Chr3-0669 (SCS7), PP7435_Chr3-0636(CRD1), PP7435_Chr3-0950 (SLC4), PP7435_Chr2-0585 (PIS1),PP7435_chr1-0934 (PRY2), ARV1 (ORF not annotated; located betweenPP7435_Chr4-0493/0494), PP7435_Chr3-0741 (ARE2), PP7435_Chr4-0963(FAD12), PP7435_Chr1-0794 (PSD1), PP7435_Chr1-0160 (SLC1),PP7435_CHR1-0078 (GPT2) PP7435_Chr3-1169 (DGK1) and PP7435_Chr2-0045(CDS1) or a functional homologue thereof, wherein the functionalhomologue has at least 30% sequence identity to an amino acid sequenceas shown in any one of SEQ ID NOs: 1-15, PP7435_Chr3-0788 (SUR2),PP7435_Chr3-1005 (PHS1), PP7435_Chr2-0350 (AUR1), PP7435_Chr4-0626(IFA38), PP7435_Chr3-0669 (SCS7), PP7435_Chr3-0636 (CRD1),PP7435_Chr3-0950 (SLC4), PP7435_Chr2-0585 (PIS1), PP7435_chr1-0934(PRY2), ARV1 (ORF not annotated; located between PP7435_Chr4-0493/0494),PP7435_Chr3-0741 (ARE2), PP7435_Chr4-0963 (FAD12), PP7435_Chr1-0794(PSD1), PP7435_Chr1-0160 (SLC1), PP7435_CHR1-0078 (GPT2)PP7435_Chr3-1169 (DGK1) and PP7435_Chr2-0045 (CDS1), respectively.

The term “protein of interest” (POI) as used herein refers to a proteinthat is produced by means of recombinant technology in a host cell. Morespecifically, the protein may either be a polypeptide not naturallyoccurring in the host cell, i.e. a heterologous protein, or else may benative to the host cell, i.e. a homologous protein to the host cell, butis produced, for example, by transformation with a self-replicatingvector containing the nucleic acid sequence encoding the POI, or uponintegration by recombinant techniques of one or more copies of thenucleic acid sequence encoding the POI into the genome of the host cell,or by recombinant modification of one or more regulatory sequencescontrolling the expression of the gene encoding the POI, e.g. of thepromoter sequence. In general, the proteins of interest referred toherein may be produced by methods of recombinant expression well knownto a person skilled in the art.

There is no limitation with respect to the protein of interest (POI).The POI is usually a eukaryotic or prokaryotic polypeptide, variant orderivative thereof. The POI can be any eukaryotic or prokaryoticprotein. Examples of POIs are described in Schmidt, Appl. Microbiol.Biotechnol. (2004), 65: 363-372 or in Kirk et al., Curr. Opin.Biotechnol. (2002), 13: 345-351. Any of the proteins mentioned in Tables1 and 2 of Schmidt and in Table 1 of Kirk et al. is encompassed by theterm “POI” as used herein. The protein can be a naturally secretedprotein or an intracellular protein, i.e. a protein which is notnaturally secreted. The present invention also includes biologicallyactive fragments of proteins. In another embodiment, a POI may be anamino acid chain or present in a complex, such as a dimer, trimer,hetero-dimer, multimer or oligomer.

The protein of interest may be a protein used as nutritional, dietary,digestive, supplements, such as in food products, feed products, orcosmetic products. The food products may be, for example, bouillon,desserts, cereal bars, confectionery, sports drinks, dietary products orother nutrition products. Preferably, the protein of interest is a foodadditive.

In another embodiment, the protein of interest may be used in animalfeeds. The POI may be a detoxifying enzyme such as a mycotoxin degradingenzyme. Mycotoxin degrading enzymes include aflatoxin detoxizyme,zearalenone esterases, zearalenone lactonases, zearalenone hydrolase,fumonisin carboxylesterases, fumonisin aminotransferases, aminopolyolamine oxidases, deoxynivalenol expoxide hydrolases. The POI may also bean enzyme which degrades ochratoxin derivatives or ergot alkaloid. Thesecompounds are toxic to living organisms including humans and farmanimals. Examples of such enzymes include ochratoxin amidase, ergotaminehydrolase, ergotamine amylase. Mycotoxin degrading enzymes in animalfeed is useful in controlling mycotoxin contamination of feed.

Further examples of POI include anti-microbial proteins, such aslactoferrin, lysozyme, lactoferricin, lactohedrin, kappa-casein,haptocorrin, lactoperoxidase, a milk protein, acute-phase proteins,e.g., proteins that are produced normally in production animals inresponse to infection.

Examples of enzymes which can be used as feed additive include phytase,xylanase and β-glucanase. A “food” means any natural or artificial dietmeal or the like or components of such meals intended or suitable forbeing eaten, taken in, digested, by a human being.

Examples of enzymes which can be used as food additive include protease,lipase, lactase, pectin methyl esterase, pectinase, transglutaminase,amylase, β-glucanase, acetolactate decarboxylase and laccase.

Enzyme

A POI may be an enzyme. Preferred enzymes are those which can be usedfor industrial application, such as in the manufacturing of a detergent,starch, fuel, textile, pulp and paper, oil, personal care products, orsuch as for baking, organic synthesis, and the like. (see Kirk et al.,Current Opinion in Biotechnology (2002) 13:345-351)

Therapeutic Protein

A POI may be a therapeutic protein. A POI may be but is not limited to aprotein suitable as a biopharmaceutical substance like an antibody orantibody fragment, or antibody derived scaffold, single domainantibodies and derivatives thereof other not antibody derived affinityscaffolds, growth factor, hormone, enzyme, vaccine, etc. as described inmore detail herein.

Preferably, the protein of interest is a mammalian polypeptide or evenmore preferably a human polypeptide. Especially preferred therapeuticproteins, which refer to any polypeptide, protein, protein variant,fusion protein and/or fragment thereof which may be administered to amammal. It is envisioned but not required that therapeutic proteinaccording to the present invention is heterologous to the cell. Examplesof proteins that can be produced by the cell of the present inventionare, without limitation, enzymes, regulatory proteins, receptors,peptide hormones, growth factors, cytokines, scaffold binding proteins(e.g. anticalins), structural proteins, lymphokines, adhesion molecules,receptors, and any other polypeptides that can serve as agonists orantagonists and/or have therapeutic or diagnostic use. Moreover, theproteins of interest may be antigens as used for vaccination, vaccines,antigen-binding proteins, immune stimulatory proteins.

Such therapeutic proteins include, but are not limited to, insulin,insulin-like growth factor, hGH, tPA, cytokines, e.g. interleukines suchas IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha,IFN beta, IFN gamma, IFN omega or IFN tau, tumor necrosisfactor (TNF)TNF alpha and TNF beta, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.

In a preferred embodiment, the protein is an antibody. The term“antibody” is intended to include any polypeptide chain-containingmolecular structure with a specific shape that fits to and recognizes anepitope, where one or more non-covalent binding interactions stabilizethe complex between the molecular structure and the epitope. Thearchetypal antibody molecule is the immunoglobulin, and all types ofimmunoglobulins, IgG, IgM, IgA, IgE, IgD, IgY, etc., from all sources,e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammals,chicken, other avians, etc., are considered to be “antibodies.” Forexample, an antibody fragment may include but not limited to Fv (amolecule comprising the VL and VH), single-chain Fv (scFV) (a moleculecomprising the VL and VH connected with by peptide linker), Fab, Fab′,F(ab′)₂, single domain antibody (sdAb) (molecules comprising a singlevariable domain and 3 CDR), and multivalent presentations thereof. Theantibody or fragments thereof may be murine, human, humanized orchimeric antibody or fragments thereof. Examples of therapeutic proteinsinclude an antibody, polyclonal antibody, monoclonal antibody,recombinant antibody, antibody fragments, such as Fab′, F(ab′)2, Fv,scFv, di-scFvs, bi-scFvs, tandem scFvs, bispecific tandem scFvs, sdAb,nanobodies, VH, and VL, or human antibody, humanized antibody, chimericantibody, IgA antibody, IgD antibody, IgE antibody, IgG antibody, IgMantibody, intrabody, diabody, tetrabody, minibody or monobody.

Further examples of therapeutic proteins include blood coagulationfactors (VII, VIII, IX), alkaline protease from Fusarium, calcitonin,CD4 receptor darbepoetin, DNase (cystic fibrosis), erythropoetin,eutropin (human growth hormone derivative), follicle stimulating hormone(follitropin), gelatin, glucagon, glucocerebrosidase (Gaucher disease),glucosamylase from A. niger, glucose oxidase from A. niger,gonadotropin, growth factors (GCSF, GMCSF), growth hormones(somatotropines), hepatitis B vaccine, hirudin, human antibody fragment,human apolipoprotein Al, human calcitonin precursor, human collagenaseIV, human epidermal growth factor, human insulin-like growth factor,human interleukin 6, human laminin, human proapolipoprotein Al, humanserum albumin, insulin, insulin and muteins, insulin, interferon alphaand muteins, interferon beta, interferon gamma (mutein), interleukin 2,luteinization hormone, monoclonal antibody 5T4, mouse collagen, OP-1(osteogenic, neuroprotective factor), oprelvekin (interleukin11-agonist), organophosphohydrolase, PDGF-agonist, phytase, plateletderived growth factor (PDGF), recombinant plasminogen-activator G,staphylokinase, stem cell factor, tetanus toxin fragment C, tissueplasminogen-activator, and tumor necrosis factor (see Schmidt, ApplMicrobiol Biotechnol (2004) 65:363-372).

Leader Sequence

The protein of interest may be linked with a leader sequence whichcauses secretion of the POI from the host cell. The presence of such asecretion leader sequence in the expression vector is required when thePOI intended for recombinant expression and secretion is a protein whichis not naturally secreted and therefore lacks a natural secretion leadersequence, or its nucleotide sequence has been cloned without its naturalsecretion leader sequence. In general, any secretion leader sequenceeffective to cause secretion of the POI from the host cell may be usedin the present invention. The secretion leader sequence may originatefrom yeast source, e.g. from yeast α-factor such as MFa of Saccharomycescerevisiae, or yeast phosphatase, from mammalian or plant source, orothers. The selection of the appropriate secretion leader sequence isapparent to a skilled person. Alternatively, the secretion leadersequence can be fused to the nucleotide sequence encoding a POI intendedfor recombinant expression by conventional cloning techniques known to askilled person prior to cloning of the nucleotide sequence in theexpression vector or the nucleotide sequence encoding a POI comprising anatural secretion leader sequence is cloned in the expression vector. Inthese cases the presence of a secretion leader sequence in theexpression vector is not required.

The recombinant nucleotide sequence(s) encoding the POI(s), as well asthose encoding the helper proteins, may also be provided on one or moreautonomously replicating plasmids in a single copy or in multiple copiesper cell.

Alternatively, the recombinant nucleotide sequence encoding the POI andthe recombinant nucleotide sequence encoding a helper protein arepresent on the same plasmid in single copy or multiple copies per cell.

Underexpression of Helper Proteins

The inventors have also identified several helper proteins (KO helperproteins) involved in lipid storage whose expression was observed tohave a negative impact on the yield of POI from a host cell. Such helperproteins a negative impact on the yield of POI from a host cell arepreferably not transcription factors. Preferred are helper proteincomprising an amino acid sequence shown in any one of SEQ ID NOs: 16 or17, or functional homologues thereof, A functional homologue of a helperprotein comprising an amino acid sequence shown in any one of SEQ IDNOs: 16 or 17 has at least 30%, such as at least 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence identity to anamino acid sequence shown in any one of SEQ ID NOs: 16 or 17.Furthermore, it has been discovered that a modification of the genesencoding the KO helper proteins of SEQ ID NOs: 16 or 17 such as mutationor deletion is able to increase the yield of POI. This disclosureprovides methods and materials useful for further improving the yield ofPOI by engineering host cells such that they underexpress the KO helpergenes identified by the inventors. If such a helper protein is presentin the host cell, it can be modified, e.g. mutated or knocked-out toimprove the POI yield. The presence of such a KO helper protein can beidentified with any method known to the art in view of the geneidentifiers or nucleotide sequences provided herein. The KO helperproteins that are advantageously absent from a host cell in order toimprove the yield of a non-membrane protein of interest are listed inTable 1 above.

Preferably, the host cell may be engineered to underexpress apolynucleotide encoding a KO protein comprising an amino acid sequenceas shown in SEQ ID NO: 16 or 17 or a functional homologue thereof,wherein the functional homologue has at least 30% sequence identity toan amino acid sequence as shown in SEQ ID NO: 16 or 17. For example, thehost cell may be engineered to underexpress a polynucleotide encoding aprotein comprising an amino acid having at least 30% sequence identityto an amino acid sequence as shown in SEQ ID NO: 16. For example, thehost cell may be engineered to underexpress a polynucleotide encoding aprotein comprising an amino acid having at least 30% sequence identityto an amino acid sequence as shown in SEQ ID NO: 17.

Preferably, when KO1 (helper protein comprising an amino acid sequenceshown in SEQ ID NO: 16) and/or KO2 (helper protein comprising an aminoacid sequence shown in SEQ ID NO: 17) is/are underexpressed, the yieldof the model protein SDZ-Fab or HyHEL-Fab in the host cell may beincreased by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290 or at least 300% and preferably by at least 10%,compared to the host cell prior to the engineering to underexpress theKO protein.

The term “underexpress” generally refers to any amount less than anexpression level exhibited by a reference standard, wherein thereference standard is the host cell prior to the engineering tounderexpress the KO protein. The terms “underexpress,”“underexpressing,” “underexpressed” and “underexpression” in the presentinvention refer an expression of a gene product or a polypeptide at alevel less than the expression of the same gene product or polypeptideprior to a genetic alteration of the host cell or in a comparable hostwhich has not been genetically altered. For example the KO protein maybeunderexpressed compared to the reference standard by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or may be even not expressedat all (reduced by 100%). No expression of the gene product or apolypeptide is also encompassed by the term “underexpression.”

Underexpression can be carried out by any method that prevents thefunctional expression of one or more of KO1 and KO2 or functionalhomologues thereof. This results in the incapability to exert itsfunction or full function. Means of underexpression may include genesilencing (e.g. RNAi genes antisense), knocking-out, altering expressionlevel, altering expression pattern, by mutagenizing the gene sequence,disrupting the sequence, insertions, additions, mutations, modifyingexpression control sequences, and the like.

Preferably, underexpression is achieved by knocking-out thepolynucleotide encoding the KO protein in the host cell. A gene can beknocked out by deleting the entire or partial coding sequence. Methodsof making gene knockouts are known in the art, e.g., see Kuhn and Wurst(Eds.) Gene Knockout Protocols (Methods in Molecular Biology) HumanaPress (Mar. 27, 2009). A gene can also be knocked out by removing partor all of the gene sequence. Alternatively, a gene can be knocked-out orinactivated by the insertion of a nucleotide sequence, such as aresistance gene. Alternatively, a gene can be knocked-out or inactivatedby inactivating its promoter. Accordingly, with respect tounderexpression of KO1 and KO2 a preferred host cell expresses SEQ IDNO: 16 or 17 or a functional homologue thereof, wherein the functionalhomologue has at least 30% sequence identity to an amino acid sequenceas shown in SEQ ID NO: 16 or 17, a fungal host cell such as Pichiapastoris, Hansenula polymorpha, Trichoderma reesei, Saccharomycescerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichiamethanolica, Candida boidinii, and Komagataella, and Schizosaccharomycespombe being preferred. Even more preferred is a fungal host cell fromthe genus Pichia pastoris, expressing SEQ ID NO: 16 or 17.

In an embodiment, underexpression is achieved by disrupting thepolynucleotide representing the gene coding for said KO protein in thehost cell.

A “disruption” is a change in a nucleotide or amino acid sequence, whichresulted in the addition, deleting, or substitution of one or morenucleotides or amino acid residues, as compared to the original sequenceprior to the disruption.

An “insertion” or “addition” is a change in a nucleic acid or amino acidsequence in which one or more nucleotides or amino acid residues havebeen added as compared to the original sequence prior to the disruption.

A “deletion” is defined as a change in either nucleotide or amino acidsequence in which one or more nucleotides or amino acid residues,respectively, have been removed (i.e., are absent). A deletionencompasses deletion of the entire sequence, deletion of part of thecoding sequence, or deletion of single nucleotides or amino acidresidue.

A “substitution” generally refers to replacement of nucleotides or aminoacid residues with other nucleotides or amino acid residues.“Substitution” for example can be performed by site-directed mutation,generation of random mutations, and gapped-duplex approaches (See e.g.,U.S. Pat. No. 4,760,025; Moring et al., Biotech. (1984) 2:646 and Krameret al., Nucleic Acids Res., (1984) 12:9441).

Preferably, disruption results in a frame shift mutation, early stopcodon, point mutations of critical residues, translation of a nonsenseor otherwise non-functional protein product.

In another embodiment, underexpression is achieved by disrupting thepromoter which is operably linked with said polypeptide to be knockedout. A promoter directs the transcription of a downstream gene. Thepromoter is necessary, together with other expression control sequencessuch as enhancers, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences, to express a given gene. Therefore, it is alsopossible to disrupt any of the expression control sequence to hinder theexpression of the polypeptide.

In another embodiment, underexpression is achieved bypost-transcriptional gene silencing (PTGS). A technique commonly used inthe art, PTGS reduces the expression level of a gene via expression of aheterologous RNA sequence, frequently antisense to the gene requiringdisruption (Lechtreck et al., J. Cell Sci (2002). 115:1511-1522; Smithet al., Nature (2000). 407:319-320; Furhmann et al., J. Cell Sci (2001).114:3857-3863; Rohr et al., Plant J (2004). 40(4):611-21.

“Underexpression” can be achieved with any known techniques in the artwhich lowers gene expression. For example, the promoter which isoperably linked with the polypeptide can be replaced with anotherpromoter which has lower promoter activity. Promoter activity may beassessed by its transcriptional efficiency. This may be determineddirectly by measurement of the amount of mRNA transcription from thepromoter, e.g. by Northern Blotting, quantitative PCR or indirectly bymeasurement of the amount of gene product expressed from the promoter.Underexpression may in another embodiment achieved by intervening in thefolding of the expressed KO protein so that the KO protein is notproperly folded to become functional. For example, mutation can beintroduced to remove a disulfide bond formation of the KO protein or todisruption the formation of an alpha helices and beta sheets.

In a further embodiment a host cell that underexpresses helper proteinKO1 and/or KO2 can overexpress one or more helper proteins of theinvention. Specific combinations of underexpressed and overexpressedhelper proteins are disclosed in the items herein.

Use

The present invention further provides the use of the engineered hostcells for manufacturing a protein of interest. The host cells can beadvantageously used for introducing polypeptides encoding one or morePOI(s), and thereafter can be cultured under suitable conditions toexpress the POI. Details of such use are described herein in the sectionconcerning methods of the present invention.

Polynucleotides encoding the helper proteins and the POI may berecombined in to the host cell by ligating the relevant genes each intoone vector. It is possible to construct single vectors carrying thegenes, or two separate vectors, one to carry the helper protein genesand the other one the POI genes. These genes can be integrated into thehost cell genome by transforming the host cell using such vector orvectors. In some embodiments, the genes encoding the POI is integratedin the genome and the gene encoding the helper protein is integrated ina plasmid or vector. In some embodiments, the genes encoding the helperprotein is integrated in the genome and the gene encoding the POI isintegrated in a plasmid or vector. In some embodiments, the genesencoding the POI and the helper protein are integrated in the genome. Insome embodiments, the gene encoding the POI and the helper protein isintegrated in a plasmid or vector. If multiple genes encoding the POIare used, some genes encoding the POI are integrated in the genome whileothers are integrated in the same or different plasmids or vectors. Ifmultiple genes encoding the helper proteins are used, some of the genesencoding the helper protein are integrated in the genome while othersare integrated in the same or different plasmids or vectors. Moreteaching ca be found in the following sections of the application.

Generally, proteins of interest can be produced using the recombinanthost cell by culturing the host cell in an appropriate medium, isolatingthe expressed POI from the culture, and purifying it by a methodappropriate for the expressed product, in particular to separate the POIfrom the cell.

The present invention further provides the use of an isolatedpolypeptide comprising an amino acid sequence having at least 30%identity to an amino acid sequence shown in any one of SEQ ID NOs: 1-15and 18 for manufacturing a protein of interest.

Method

The present invention further relates to a method of increasing theyield of a protein of interest in a host cell, comprising overexpressinga polynucleotide of the present invention. The polynucleotide encodes ahelper protein comprising an amino acid having at least 30% sequenceidentity to an amino acid sequence as shown in any one of SEQ ID NOs:1-15.

The present invention further relates to a method of increasing theyield of a protein of interest in a host cell, comprisingunderexpressing a helper protein comprising an amino acid having atleast 30% sequence identity to an amino acid sequence as shown in SEQ IDNO: 16 and/or 17.

As used herein, the term “increasing the yield of a protein of interestin a host cell” means that the yield of the protein of interest isincreased when compared to the same cell expressing the same POI underthe same culturing conditions, however, without the polynucleotideencoding the helper protein being overexpressed.

As will be appreciated by a skilled person in the art, theoverexpression of the helper proteins of the present invention have beenshown to increase product yield of POI. Therefore, for a given host cellwhich expressed a POI with a level that should be increased, it ispossible to apply the present invention by expressing any one or severalof the helper proteins in the host cell, if helper protein is notpresent in the host cell, or further increasing the level of expressionthe helper proteins in the cell, if genes encoding the helper protein isalready present in the host cell.

The present invention further provides a method of increasing the yieldof a protein of interest in a host cell. The method comprises (i)engineering the host cell to express or overexpress a helper protein,(ii) engineering said host cell to comprise a heterologouspolynucleotide encoding said protein of interest, and (iii) culturingsaid host cell under suitable conditions to overexpress the helperprotein and to express the protein of interest and optionally (iv)isolating the protein of interest from the cell culture. It should benoted that the steps recited in (i) and (ii) does not have to beperformed in the recited sequence. It is possible to first perform thestep recited in (ii) and then (i). In step (i), the host cell can beengineered to overexpress a polynucleotide encoding a helper proteincomprising an amino acid having at least 30% sequence identity to anamino acid sequence as shown in any one of SEQ ID NOs: 1-15.

The present invention further provides a method of increasing the yieldof a protein of interest in a host cell. The method comprises (i)engineering the host cell to underexpress a helper protein, (ii)engineering said host cell to comprise a heterologous polynucleotideencoding said protein of interest, and (iii) culturing said host cellunder suitable conditions to express the protein of interest andoptionally (iv) isolating the protein of interest from the cell culture.It should be noted that the steps recited in (i) and (ii) do not have tobe performed in the recited sequence. It is possible to first performthe step recited in (ii) and then (i). In step (i), the host cell can beengineered to underexpress a polynucleotide encoding a helper proteincomprising an amino acid having at least 30% sequence identity to anamino acid sequence as shown in SEQ ID NO: 16 and 17.

Procedures used to manipulate polynucleotide sequences, e.g. coding forthe helper proteins and/or the POI, the promoters, enhancers, leaders,etc., are well known to persons skilled in the art, e.g. described by J.Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition),Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NewYork (2001).

A foreign or target polynucleotide such as the polynucleotdies encodingthe overexpressed helper protein or POI can be inserted into thechromosome by various means, e.g., by homologous recombination or byusing a hybrid recombinase that specifically targets sequences at theintegration sites. The foreign or target polynucleotide described aboveis typically present in a vector (“inserting vector”). These vectors aretypically circular and linearized before used for homologousrecombination. As an alternative, the foreign or target polynucleotidesmay be DNA fragments joined by fusion PCR or synthetically constructedDNA fragments which are then recombined into the host cell. In additionto the homology arms, the vectors may also contain markers suitable forselection or screening, an origin of replication, and other elements. Itis also possible to use heterologous recombination which results inrandom or non-targeted integration. Heterologous recombination refers torecombination between DNA molecules with significantly differentsequences. Methods of recombinations are known in the art and forexample described in Boer et al., Appl Microbiol Biotechnol (2007)77:513-523. One may also refer to Principles of Gene Manipulation andGenomics by Primrose and Twyman (7^(th) edition, Blackwell Publishing2006) for genetic manipulation of yeast cells.

Polynucleotides encoding the overexpressed helper protein and/or POI mayalso be present on an expression vector. Such vectors are known in theart and already described above. In expression vectors, a promoter isplaced upstream of the gene encoding the heterologous protein andregulates the expression of the gene. Multi-cloning vectors areespecially useful due to their multi-cloning site. For expression, apromoter is generally placed upstream of the multi-cloning site. Avector for integration of the polynucleotide encoding a helper proteinand/or the POI may be constructed either by first preparing a DNAconstruct containing the entire DNA sequence coding for the helperprotein and/or the POI and subsequently inserting this construct into asuitable expression vector, or by sequentially inserting DNA fragmentscontaining genetic information for the individual elements, such as theleader sequence, the target DNA sequence, followed by ligation. As analternative to restriction and ligation of fragments, recombinationmethods based on attachment sites (att) and recombination enzymes may beused to insert DNA sequences into a vector. Such methods are described,for example, by Landy (1989) Ann. Rev. Biochem. 58:913-949; and areknown to those of skill in the art.

Host cells according to the present invention can be obtained byintroducing a vector or plasmid comprising the target polynucleotidesequences into the cells. Techniques for transfecting or transformingeukaryotic cells or transforming prokaryotic cells are well known in theart. These can include lipid vesicle mediated uptake, heat shockmediated uptake, calcium phosphate mediated transfection (calciumphosphate/DNA co-precipitation), viral infection, particularly usingmodified viruses such as, for example, modified adenoviruses,microinjection and electroporation. For prokaryotic transformation,techniques can include heat shock mediated uptake, bacterial protoplastfusion with intact cells, microinjection and electroporation. Techniquesfor plant transformation include Agrobacterium mediated transfer, suchas by A. tumefaciens, rapidly propelled tungsten or goldmicroprojectiles, electroporation, microinjection and polyethylyneglycol mediated uptake. The DNA can be single or double stranded, linearor circular, relaxed or supercoiled DNA. For various techniques fortransfecting mammalian cells, see, for example, Keown et al. (1990)Processes in Enzymology 185:527-537.

The present invention further provides a method of manufacturing aprotein of interest in a host cell comprising (i) providing the hostcell engineered to overexpress a polynucleotide encoding a helperprotein comprising an amino acid sequence as shown in any one of SEQ IDNOs: 1-15 or a functional homologue thereof, wherein the functionalhomologue has at least 30% sequence identity to an amino acid sequenceas shown in any one of SEQ ID NOs: 1-15, wherein said host cellcomprises a heterologous polynucleotide encoding a protein of interest;and (ii) culturing the host cell under suitable conditions tooverexpress the helper protein or functional homologue thereof and toexpress the protein of interest and optionally isolating the protein ofinterest from the cell culture.

The present invention further provides a method of manufacturing aprotein of interest in a host cell comprising (i) providing the hostcell engineered to underexpress a polynucleotide encoding a helperprotein comprising an amino acid as shown in any one of SEQ ID NOs: 16or 17 or a functional homologue thereof, wherein the functionalhomologue has at least 30% sequence identity to an amino acid sequenceas shown in SEQ ID NO: 16 or 17 wherein said host cell comprises aheterologous polynucleotide encoding a protein of interest; and (ii)culturing the host cell under suitable conditions to express protein ofinterest and optionally isolating the protein of interest. Preferably,the host cell is engineered to underexpress both polynucleotidesencoding a helper protein comprising an amino acid as shown in any oneof SEQ ID NOs: 16 or 17 or a functional homologue thereof.

It is understood that the methods disclosed herein may further includecultivating said recombinant host cells under conditions permitting theexpression of the POI and helper protein. A recombinantly produced POIcan then be isolated from the cell or the cell culture medium, dependingon the nature of the expression system and the expressed protein, e.g.whether the protein is fused to a signal peptide and whether the proteinis soluble or non-soluble. As will be understood by the skilled artisan,cultivation conditions will vary according to factors that include thetype of host cell, in particular the expression vector employed. Signalpeptides generally contain a positively charged N-terminus followed by ahydrophobic core, followed by a recognition site for an enzyme known assignal peptidase. This enzyme cleaves the signal peptide from theprotein during translocation. The protein is transported from theendoplasmic reticulum to the Golgi apparatus, and then follows one of anumber of routes in the secretory pathway, depending on the nature ofthe protein. The protein may be secreted into the culture medium or maybe retained in the cell, for example. The leader sequences of certainsecreted proteins comprise peptides that are located C-terminal to thesignal peptide and are processed from the mature protein of interestsubsequent to cleavage of the signal peptide. Such leaders often arereferred to as prepro peptides, wherein the pre region is the signalsequence and the pro region designates the remainder of the leader.

One example is the yeast α-factor leader, which contains a signalpeptide (including a C-terminal signal peptidase recognition site(Ala-Leu-Ala) followed by a pro region containing a basic amino acidpair (Lys-Arg) that constitutes a KEX2 protease processing site,immediately followed by the peptide Glu-Ala-Glu-Ala at the C-terminus ofthe pro region. Processing of this leader involves removal of the signalpeptide by signal peptidase, followed by cleavage between the Lys andArg residues by KEX2 protease. The GluAlaGluAla residues aresubsequently removed by a peptidase that is the product of the STE13gene (Julius et al., Cell (1983) 32:839). The yeast α-factor leader isdescribed in U.S. Pat. No. 4,546,082. Signal peptides derived fromproteins naturally secreted by yeast cells have been employed inrecombinant expression systems for production of heterologous proteinsin yeast. The use of mammalian signal peptides in yeast expressionsystems also has been reported, although certain of the mammalian signalpeptides were not effective in promoting secretion of heterologousproteins in yeast.

The phrase “culturing under suitable condition such that a desiredpolypeptide is expressed” refers to maintaining and/or growingmicroorganisms under conditions (e.g., temperature, pressure, pH,induction, growth rate, medium, duration, etc.) appropriate orsufficient to obtain production of the desired compound or to obtaindesired polypeptide.

A host cell according to the invention obtained by transformation withthe helper protein gene(s), and/or the POI genes or by underexpressingthe helper protein gene(s) and/or by transformation the POI genes maypreferably first be cultivated at conditions to grow efficiently to alarge cell number without the burden of expressing a heterologousprotein. When the cells are prepared for POI expression, suitablecultivation conditions are selected and optimized to produce the POI.

By way of example, using different promoters and/or copies and/orintegration sites for the helper gene(s) and the POI(s), the expressionof the helper genes can be controlled with respect to time point andstrength of induction in relation to the expression of the POI(s). Forexample, prior to induction of POI expression, the helper protein(s) maybe first expressed. This has the advantage that the helper proteinsis/are already present at the beginning of POI translation.Alternatively, the helper protein(s) and POI(s) can be induced at thesame time. In another example, prior to induction of POI expression, thehelper protein(s) may be first underexpressed. This has the advantagethat the helper proteins is/are already absent at the beginning of POItranslation.

An inducible promoter may be used that becomes activated as soon as aninductive stimulus is applied, to direct transcription of the gene underits control. Under growth conditions with an inductive stimulus, thecells usually grow more slowly than under normal conditions, but sincethe culture has already grown to a high cell number in the previousstage, the culture system as a whole produces a large amount of theheterologous protein. An inductive stimulus is preferably the additionof an appropriate agents (e.g. methanol for the AOX-promoter) or thedepletion of an appropriate nutrient (e.g., methionine for theMET3-promoter). Also, the addition of ethanol, methylamine, cadmium orcopper as well as heat or an osmotic pressure increasing agent caninduce the expression depending on the promotors operably linked to thehelper gene(s) and the POI(s).

It is preferred to cultivate the hosts according to the invention in abioreactor under optimized growth conditions to obtain a cell density ofat least 1 g/L, preferably at least 10 g/L cell dry weight, morepreferably at least 50 g/L cell dry weight. It is advantageous toachieve such yields of biomolecule production not only on a laboratoryscale, but also on a pilot or industrial scale.

According to the present invention, due to overexpression of the helperproteins, and/or underexpression of KO proteins, the POI is obtainablein high yields, even when the biomass is kept low. Thus, a high specificyield, which is measured in mg POI/g dry biomass, may be in the range of1 to 200, such as 50 to 200, such as 100-200, in the laboratory, pilotand industrial scale is feasible. The specific yield of a productionhost according to the invention preferably provides for an increase ofat least 1.1 fold, more preferably at least 1.2 fold, at least 1.3 or atleast 1.4 fold, in some cases an increase of more than 2 fold can beshown, when compared to the expression of the product without theoverexpression of helper proteins and/or underexpression of KO proteins.

The host cell according to the invention may be tested for itsexpression/secretion capacity or yield by standard tests, e.g. ELISA,activity assays, HPLC, Surface Plasmon Resonance (Biacore), WesternBlot, capillary electrophoresis (Caliper) or SDS-Page.

Preferably, the cells are cultivated in a minimal medium with a suitablecarbon source, thereby further simplifying the isolation processsignificantly. By way of example, the minimal medium contains anutilizable carbon source (e.g. glucose, glycerol, ethanol or methanol),salts containing the macro elements (potassium, magnesium, calcium,ammonium, chloride, sulphate, phosphate) and trace elements (copper,iodide, manganese, molybdate, cobalt, zinc, and iron salts, and boricacid).

In the case of yeast cells, the cells may be transformed with one ormore of the above-described expression vector(s), mated to form diploidstrains, and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants oramplifying the genes encoding the desired sequences. A number of minimalmedia suitable for the growth of yeast are known in the art. Any ofthese media may be supplemented as necessary with salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES,citric acid and phosphate buffer), nucleosides (such as adenosine andthymidine), antibiotics, trace elements, vitamins, and glucose or anequivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression and are known to the ordinarily skilled artisan. Cell cultureconditions for other type of host cells are also known and can bereadily determined by the artisan. Descriptions of culture media forvarious microorganisms are for example contained in the handbook “Manualof Methods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

Cells can be cultured (e.g., maintained and/or grown) in liquid mediaand preferably are cultured, either continuously or intermittently, byconventional culturing methods such as standing culture, test tubeculture, shaking culture (e.g., rotary shaking culture, shake flaskculture, etc.), aeration spinner culture, or fermentation. In someembodiments, cells are cultured in shake flasks or deep well plates. Inyet other embodiments, cells are cultured in a bioreactor (e.g., in abioreactor cultivation process). Cultivation processes include, but arenot limited to, batch, fed-batch and continuous methods of cultivation.The terms “batch process” and “batch cultivation” refer to a closedsystem in which the composition of media, nutrients, supplementaladditives and the like is set at the beginning of the cultivation andnot subject to alteration during the cultivation; however, attempts maybe made to control such factors as pH and oxygen concentration toprevent excess media acidification and/or cell death. The terms“fed-batch process” and “fed-batch cultivation” refer to a batchcultivation with the exception that one or more substrates orsupplements are added (e.g., added in increments or continuously) as thecultivation progresses. The terms “continuous process” and “continuouscultivation” refer to a system in which a defined cultivation media isadded continuously to a bioreactor and an equal amount of used or“conditioned” media is simultaneously removed, for example, for recoveryof the desired product. A variety of such processes has been developedand is well-known in the art.

In some embodiments, cells are cultured for about 12 to 24 hours, inother embodiments, cells are cultured for about 24 to 36 hours, about 36to 48 hours, about 48 to 72 hours, about 72 to 96 hours, about 96 to 120hours, about 120 to 144 hours, or for a duration greater than 144 hours.In yet other embodiments, culturing is continued for a time sufficientto reach desirable production yields of POI.

The above mentioned methods may further comprise a step of isolating theexpressed POI. If the POI is secreted from the cells, it can be isolatedand purified from the culture medium using state of the art techniques.Secretion of the POI from the cells is generally preferred, since theproducts are recovered from the culture supernatant rather than from thecomplex mixture of proteins that results when cells are disrupted torelease intracellular proteins. A protease inhibitor, such as phenylmethyl sulfonyl fluoride (PMSF) may be useful to inhibit proteolyticdegradation during purification, and antibiotics may be included toprevent the growth of adventitious contaminants. The composition may beconcentrated, filtered, dialyzed, etc., using methods known in the art.Alternatively, cultured host cells may also be ruptured sonically ormechanically, enzymatically or chemically to obtain a cell extractcontaining the desired POI, from which the POI may be isolated andpurified.

As isolation and purification methods for obtaining the POI may be basedon methods utilizing difference in solubility, such as salting out andsolvent precipitation, methods utilizing difference in molecular weight,such as ultrafiltration and gel electrophoresis, methods utilizingdifference in electric charge, such as ion-exchange chromatography,methods utilizing specific affinity, such as affinity chromatography,methods utilizing difference in hydrophobicity, such as reverse phasehigh performance liquid chromatography, and methods utilizing differencein isoelectric point, such as isoelectric focusing may be used. Specificpurification steps are preferably employed to remove any helper proteinthat is also expressed and would contaminate the POI preparation.

The isolated and purified POI can be identified by conventional methodssuch as Western Blotting or specific assays for POI activity. Thestructure of the purified POI can be determined by amino acid analysis,amino-terminal peptide sequencing, primary structure analysis forexample by mass spectrometry, and the like. It is preferred that the POIis obtainable in large amounts and in a high purity level, thus meetingthe necessary requirements for being used as an active ingredient inpharmaceutical compositions or as feed or food additive.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention and defined in the claims.Efforts have been made to ensure accuracy with respect to the numbersused (e.g. amounts, temperature, concentrations, etc.) but someexperimental errors and deviations should be allowed for. Unlessotherwise indicated, parts are parts by weight, molecular weight isaverage molecular weight, temperature is in degrees centigrade; andpressure is at or near atmospheric.

The examples below will demonstrate that the newly identified proteinsinvolved in lipid biosynthesis (i.e. helper protein(s) increase(s) upontheir over-respectively underexpression the titer (product per volume inmg/L) and the yield (product per biomass in mg/g biomass measured as drycell weight or wet cell weight), respectively, of a POI upon itsexpression. As an example, the yield of recombinant antibody Fabfragments and recombinant enzymes in the yeast Pichia pastoris areincreased. The positive effect was shown in shaking cultures (conductedin shake flasks or deep well plates) and in lab scale fed-batchcultivations.

Example 1: Generation of P. pastoris Production Strains

a) Construction of P. pastoris Strains Secreting Antibody Fab FragmentHyHEL

P. pastoris CBS7435 (CBS, genome sequenced by Küberl et al. 2011)mut^(S) (obtained from CBS-KNAW Fungal Biodiversity Center, Uetrecht,The Netherlands) variant was used as host strain. The pPM2d_pGAP andpPM2d_pAOX expression vectors are derivatives of the pPuzzle_ZeoR vectorbackbone described in WO2008/128701A2, consisting of the pUC19 bacterialorigin of replication and the Zeocin antibiotic resistance cassette.Expression of the heterologous gene is mediated by the P. pastorisglyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or alcoholoxidase (AOX) promoter, respectively, and the S. cerevisiae CYC1transcription terminator. The light chain (LC) (SEQ ID NO: 44) and theheavy chain (HC) (SEQ ID NO: 43) of the antibody Fab fragment HyHEL(FIG. 2 ) were amplified from vector DNA template (carrying the gene ofinterests with N-terminal S. cerevisiae alpha mating factor signalleader sequence) using the primers for HyHEL-HC and HyHEL-LC in Table 3,and each ligated into both vectors pPM2d_pGAP and pPM2d_pAOX digestedwith SbfI and SfiI. The LC fragments were ligated into variants ofpPM2d_pGAP and pPM2d_pAOX, where one restriction enzyme site in thepromoter region was exchanged for another to allow subsequentlinearization (NdeI instead of AvrII in pPM2d_pGAP, Bsu36I instead ofBpu1102I in pPM2d_pAOX), the HC fragments were ligated into theunmodified versions of the vectors. After sequence verification of LCand HC, the expression cassettes for both chains were combined onto onevector by using the compatible restriction enzymes MreI and AgeI.

Plasmids were linearized using NdeI restriction enzyme (for pPM2d_pGAP)or Bsu36I restriction enzyme (for pPM2d_pAOX), respectively, prior toelectroporation (using a standard transformation protocol described inGasser et al. 2013. Future Microbiol. 8(2):191-208) into P. pastoris.Selection of positive transformants was performed on YPD plates (perliter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar-agar)containing 50 μg/mL of Zeocin. Colony PCR was used to ensure thepresence of the transformed plasmid. Therefore, genomic DNA was obtainedby cooking and freezing of P. pastoris colonies for 5 minutes each anddirectly applied for PCR with the appropriate primers. The resultingstrains were named CBS7435 mutS pAOX HyHEL-Fab or CBS7435 mutS pGAPHyHEL-Fab, respectively.

b) Construction of a P. pastoris Strain Secreting Antibody Fab FragmentSDZ

The light chain (LC) and the heavy chain (HC) of the antibody Fabfragment SDZ (FIG. 2 ) were amplified from vector DNA template (carryingthe gene of interests with N-terminal alpha mating factor signal leadersequence) using the primers for SDZ-HC and SDZ-LC in Table 2, and eachligated into pPM2d_pAOX or the variant of pPM2d_pAOX with the Bsu36Irestriction site, respectively, each digested with SbfI and SfiI. Aftersequence verification of LC and HC, the expression cassettes for bothchains were combined onto one vector by using the compatible restrictionenzymes MreI and AgeI.

Plasmids were linearized using Bsu36I restriction enzyme prior toelectroporation (using a standard transformation protocol described inGasser et al. 2013. Future Microbiol. 8(2):191-208) into P. pastoris.Selection of positive transformants was performed on YPD plates (perliter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar-agar)containing 50 μg/mL of Zeocin. Colony PCR was used to ensure thepresence of the transformed plasmid. Therefore, genomic DNA was obtainedby cooking and freezing of P. pastoris colonies for 5 minutes each anddirectly applied for PCR with the appropriate primers. The resultingstrains were named CBS7435 mutS pAOX SDZ-Fab or CBS7435 mutS pGAPSDZ-Fab, respectively.

Table 2 shows oligonucleotide primers for PCR amplification of HyHEL LCand HC as well as SDZ LC and HC (Alpha-mating factor_forward is theforward primer for amplification of all Fab chains).

TABLE 2 Restriction site Primer attached sequence Alpha-mating SbflACTACCTGCAGGCGAAACGAT factor_forward* GAGATTCCCATC SEQ ID NO: 99HyHEL-HC Sfil TCATGGCCGAGGCGGCCCTAT backward TACTTGTCACAGGACTTTGGCTC SEQ ID NO: 100 HyHEL-LC Sfil CTATGGCCGAGGCGGCCCTAT backwardTAACACTCACCTCTGTTG SEQ ID NO: 101 SDZ-HC back Sfil TATCGGCCGAGGCGGCCCTATTACTTACCTGGGGACAAG SEQ ID NO: 102 SDZ-LC back Sfil CTATGGCCGAGGCGGCCCTATTAACACTCACCTCTGTTG SEQ ID NO: 103

Example 2: Evaluation of Engineered P. pastoris Strains

The methylotrophic yeast P. pastoris is a well accepted host for theoverexpression and production of heterologous or recombinant proteinsincluding proteins of interest (POI) already employing manifoldapplications which provide a surplus in the production/secretioncapacity. Such host strain engineering strategies include for exampleoverexpression of helper proteins such as chaperones or other componentsof the protein folding machinery (Zhang et al., Biotechnol Prog. (2006).22(4):1090-1095). Most of the applications aiming to improve P. pastorishosted recombinant protein production/secretion were not considering P.pastoris' cell biology in a broader context.

Despite its frequent utilization in biotechnological relevantapplications, the amount of cell biological information such as forexample membrane characteristics and features available on P. pastorisis poor. Therefore investigated was the effect of over- orunderexpression as defined herein of proteins involved in lipidmetabolism such as sphingolipid biosynthesis including fatty acidelongation, phospholipid biosynthesis, lipid transport, ergosterolbiosynthesis, or lipid storage alone or in combination on recombinant orheterologous protein production/secretion. As model protein for such aprotein of interest, two different Fab proteins were used. Additionally,also the overexpression of a chaperone in combination with a proteininvolved in lipid metabolism was investigated.

Example 3: Generation of Strains Overexpressing Target Genes

For the investigation of positive effects on Fab secretion, diverseproteins involved in lipid metabolism (see Table 1 herein) wereoverexpressed alone or in combination, or in combination with achaperone in two different Fab producing strains: CBS7435 pPM2d_pAOXHyHEL and CBS7435 pPM2d_pAOX SDZ (generation see Example 1).

-   -   a) Amplification and cloning of the lipid metabolism or        chaperone genes into pPM2aK21 expression vectors

The genes overexpressed alone (Table 3; see results of Experiment 4 a-c)and in combination (Table 3 results of Experiment 6a) were amplified byPCR (Phusion Polymerase, ThermoFisher Scientific) from start (includinginitial 3 or 4 nucleotides of the authentic Kozak sequence) to stopcodon using the primers shown in Table 3 and 5. Genomic DNA from P.pastoris strain CBS7435 mut^(S) served as a template (Table 3) except nocorresponding coding sequence could be retrieved for wanted genes fromsequence searches. In this case, more precisely concerning LIP1 or TSC3,S. cerevisiae encoded sequences were amplified from genomic DNA from S.cerevisiae BY4741 using the primers shown in Table 3. The sequences werecloned into the MCS of the pPM2aK21 expression vector with the tworestriction enzymes SbfI and SfiI. pPMKaK21 is a derivative of pPM2d(described in Example 1a), consisting of an AOX terminator sequence (forintegration into the native AOX terminator locus), an origin ofreplication for E. coli (pUC19), an antibiotic resistance cassette(kanMX conferring resistance to Kanamycin and G418) for selection in E.coli and yeast, an expression cassette for the gene of interest (GOI),coding for the protein involved in lipid metabolism or the chaperone,comprising a GAP promoter, a multiple cloning site (MCS) and the S.cerevisiae CYC1 transcription terminator. Gene sequences were verifiedby Sanger sequencing.

TABLE 3 Gene identities of Pichia pastoris sequences areretrieved from Sturmberger et al. . [J. Biotechnol.(2016). 235(4):121-131)] and gene identities ofSaccharomyces cerevisiae sequences are retrievedfrom Cherry J.M. et al. [Nucleic Acids Res. (2012) 40 (Database issue)].Forward primer Backward primer Gene (Sbfl attached) (Sfil attached) Geneidentifier (ORF name Pichia pastoris CBS7435) ELO3 PP7435_Chr3-0987CTCGCCTGCAGGACC ACGGCCGAGGCGGCC ATGAGTGACATTAAT AGTTAGGCACGACGGACTCTGTCGC ACACTAG SEQ ID NO: 47 SEQ ID NO: 48 ELO2 PP7435_Chr3-0603TCTACCTGCAGGAAC TATGGCCGAGGCGGC GATGTCCATTCTCTC CTCAGGCCTTGCGAG ATTTGAGCGCAAATTTC SEQ ID NO: 49 SEQ ID NO: 50 LAG1 PP7435_Chr1-0676ATACCTGCAGGACAA ATTGGCCGAGGCGGC TGTCTAAAGAGGAAA CCTATTCTTCCTTCT AGACAAGTGGAGG SEQ ID NO: 51 SEQ ID NO: 52 LAC1 PP7435_Chr2-0202 ATACCTGCAGGACAAATTGGCCGAGGCGGC TGGGTGTTGAAACAT CTCAAGAACTCTCCT CTTC CATCATCSEQ ID NO: 53 SEQ ID NO: 54 LCB1 PP7435_Chr1-1525 ATACCTGCAGGACAAATTGGCCGAGGCGGC TGAGCCAACGTGAAG CTCAAAGCTGTTGCA SEQ ID NO: 55 AAACSEQ ID NO: 56 LCB2 PP7435_Chr3-0462 ATACCTGCAGGACAA ATTGGCCGAGGCGGCTGTCAAAAACTATCC CTTAGTACATGGCTT CAGATG TCTTGC SEQ ID NO: 57SEQ ID NO: 58 TSC13 PP7435_Chr4-0176 ATACCCTGCAGGCAC ATAGGCCGAGGCGGCCAATGGTTAAACTCA CCTACAAAAGGAATG TTG G SEQ ID NO: 59 SEQ ID NO: 60 Geneidentifier (ORF name Saccharomyces cerevisiae S288C) LIP1 YMR298WTTCCCTGCAGGACCA ACGGCCGAGGCGGCC TGTCTCAACCCACTC TCACATGTGATAAATSEQ ID NO: 61 TGTG SEQ ID NO: 62 TSC3 YBR058C-A TTCCCTGCAGGGAAAACGGCCGAGGCGGCC TGACACAACATAAAA TCAAAGGAAGCAATA GCTCGATGG CTTTAGSEQ ID NO: 63 SEQ ID NO: 64 Gene identifier (ORF name Pichia pastorisCBS7435) PAH1 PP7435_Chr3-0694 TTCCCTGCAGGACCA TATGGCCGAGGCGGCTGCAGTACGTAGGTA CTCAGCTGTCATCGA G TTC SEQ ID NO: 65 SEQ ID NO: 66 PRY1PP7435_Chr3-1160 ATACCTGCAGGACAA ATAGGCCGAGGCGGC TGAAGCTCTCCACCACGTCAAACAGGAGGC ATTTG AGGAC SEQ ID NO: 67 SEQ ID NO: 68 ERG11PP7435 Chr3-0214 TCTCCTGCAGGCAAA TATGGCCGAGGCGGC CATGAGTCTGGTCCACAACTAATTATCGCG G TCTTTC SEQ ID NO: 69 SEQ ID NO: 70 HMG1PP7435_Chr2-0242 TTCCCTGCAGGACGA AGGCCGAGGCGGCCT TGCTTACTGGGTTGTCAAGATTTAATGCAA C ATCTTAG SEQ ID NO: 71 SEQ ID NO: 72 t1HMG1^(§)PP7435_Chr2-0242 TACCCTGCAGGACGA AGGCCGAGGCGGCCT TGGGAATAAGTGCCACAAGATTTAATGCAA CAATC ATCTTAG SEQ ID NO: 73 SEQ ID NO: 74 t2HMG1^(§)PP7435_Chr2-0242 TTCCCTGCAGGACGA AGGCCGAGGCGGCCT TGACGCCAGACGTTGCAAGATTTAATGCAA TTCC ATCTTAG SEQ ID NO: 75 SEQ ID NO: 76 KAR2PP7435_Chr2-1167 ATACCTGCAGGACAA ATAGGCCGAGGCGGC TGCTGTCGTTAAAACCCTACAACTCATCAT CATC GATCATAGTC SEQ ID NO: 77 SEQ ID NO: 78^(§)DNA-Sequences of HMG1 were next to the full-length sequence used astwo truncated variants (t1, t2) which were depleted of their membraneanchoring domains that is depletion of initial 1449 nucleotides forvariant t1HMG1 or 1543 nucleotides in case of t2HMG1 (except theirstarting ATG-codon).

b) Overexpression of at Least Two Genes Encoding Proteins Involved inLipid Metabolism or a Chaperone in Fab Producing Strains

The P. pastoris Fab overproducing strains CBS7435 mut^(S) pAOX HyHEL-Faband CBS7435 mut^(S) pAOX SDZ-Fab were used as host strains foroverexpression of two, three, or four genes encoding proteins involvedin lipid metabolism or a chaperone (see Example 3a). Beforetransformation (using a standard transformation protocol described inGasser et al. 2013. Future Microbiol. 8(2):191-208) into the Fabproducing strains, the pPM2aK21 vectors containing the genes selectedfrom Table 3 are linearized in the AOX terminator sequence with therestriction enzyme AscI. Positive transformants were selected on YPDagar plates containing G418 and Zeocin.

Example 4: Screening for Fab Expression

In small-scale screenings, 8 to 12 transformants of each overexpressingone gene (see Table 1 and 3) encoding a protein involved in lipidmetabolism were tested in P. pastoris Fab production strain CBS7435mut^(S) pAOX HyHEL-Fab. Transformants were evaluated by comparison tothe parental host CBS7435 mut^(S) pAOX HyHEL-Fab which wasco-transformed with the linearized empty vector (pPM2aK21) and ranked,based on their impact on cell growth, Fab titer and Fab yield.

A selection of genes encoding a protein involved in lipid metabolismwere re-evaluated in the background of an additional host strain,CBS7435 mutS pAOX SDZ-Fab. Again, 8-12 transformants were tested incomparison to the parental host CBS7435 mut^(S) pAOX SDZ-Fab which wasco-transformed with the linearized empty vector (pPM2aK21).

a) Small Scale Cultivation of Pichia pastoris Fab Production Strains

2 mL YP-medium (10 g/L yeast extract, 20 g/L peptone) containing 10 g/Lglycerol and 50 μg/mL Zeocin were inoculated with a single colony of P.pastoris strains and grown overnight at 25° C. Aliquots of thesecultures (corresponding to a final OD600 of 2.0) were transferred to 2mL of Synthetic screening medium M2 (media composition is given below)supplemented with 20 g/L glucose and a glucose feed tablet (Kuhner,Switzerland; CAT #SMFB63319) and incubated for 25 h at 25° C. at 280 rpmin 24 deep well plates. The cultures were washed once by centrifugation,then the pellets were resuspended in Synthetic screening medium M2 andaliquots (corresponding to a final OD600 of 4.0) were transferred into 2mL of Synthetic screening medium M2 in fresh 24 deep well plates.Methanol (5 g/L) was added repeatedly every 12 h for 48 hours, beforecells were harvested by centrifugation at 2,500×g for 10 min at roomtemperature and prepared for analysis. Biomass was determined bymeasuring the cell weight of 1 mL cell suspension, while determinationof the recombinant secreted protein in the supernatant is described inthe following Examples 4b-4c.

Synthetic screening medium M2 contained per litre: 22.0 g Citric acidmonohydrate 3.15 g (NH₄)₂PO₄, 0.49 g MgSO₄*7H₂O, 0.80 g KCl, 0.0268 gCaCl₂*2H₂O, 1.47 mL PTM1 trace metals, 4 mg Biotin; pH was set to 5 withKOH (solid).

b) SDS-PAGE & Western Blot Analysis

For protein gel analysis the Bio-Rad Mini-Protean Tetra Cell system wasused, using 12.5% separation gels (Tris-based discontinuous buffersystem) and Tris-Glycin running buffer. After electrophoresis, theproteins were either visualized by colloidal Coomassie staining ortransferred to a nitrocellulose membrane for Western blot analysis.Therefore, the proteins were electroblotted onto a nitrocellulosemembrane using the Mini Trans-Blot® Cell for wet (tank) transfer(Bio-Rad) according to the manufacturer's instructions. After blocking,the Western Blots were probed with the following antibodies: For Fablight chain: anti-human kappa light chains (bound and free)—peroxidase(HRP) conjugated antibody, Sigma A7164 (1:5,000); For Fab heavy chain:Mouse Anti-Human IgG antibody (Ab7497, Abcam) diluted 1:1,000 andAnti-Mouse IgG (whole molecule)—Peroxidase conjugated antibody producedin goat (A4416, Sigma) as secondary antibody diluted 1:5,000.

Detection was performed with the chemiluminescent Super Signal WestChemiluminescent Substrate (Thermo Scientific) for HRP-conjugates.

c) Quantification of Fab by ELISA

Quantification of intact Fab by ELISA was done using anti-human IgGantibody (ab7497, Abcam) as coating antibody and a goat anti-human IgG(Fab specific)—peroxidase conjugated antibody (Sigma A0293) as detectionantibody. Human Fab/Kappa, IgG fragment (Bethyl P80-115) was used asstandard with a starting concentration of 100 ng/mL, supernatant sampleswere diluted accordingly. TMB (biomol E102) was used as substrate fordetection. Coating-, Dilution- and Washing buffer were based on PBS (2mM KH₂PO₄, 10 mM Na₂HPO₄·2 H₂O, 2.7 mM g KCl, 8 mM NaCl, pH 7.4) andcompleted with BSA (1% (w/v)) and/or Tween20 (0.1% (v/v)) accordingly.

Table 4 shows the Fold Change (FC) levels of titers and yields for themodel POI HyHEL-Fab estimated by ELISA of engineered strainsoverexpressing a gene as indicated in Table 5. The values are given asrelative numbers compared to the parental strain CBS7435 mut^(S) pAOXHyHEL-Fab overexpressing the empty integration vector. The number ofclones investigated is given in brackets (n, number of clones tested).Gene identities of Pichia pastoris sequences are retrieved fromSturmberger et al. [J. Biotechnol. (2016). 235(4):121-131)] and geneidentities of Saccharomyces cerevisiae sequences are retrieved fromCherry J. M. et al. [Nucleic Acids Res. (2012) 40 (Database issueD700-D705)].

TABLE 4 CBS7435 mut^(S) pAOX HyHEL-Fab Gene Identifier (ORF name FC ofTiter FC of Yield Gene Pichia pastoris CBS7435) average average ELO3PP7435_chr3-0987 1.80 (n = 8) 1.90 (n = 8) LAG1 PP7435_Chr1-0676 1.01 (n= 12) 1.18 (n = 12) PAH1 PP7435_chr3-0694 0.94 (n = 3) 1.43 (n = 3) PRY1PP7435_chr3-1160 1.48 (n = 9) 1.39 (n = 9) ERG11 PP7435_chr3-0214 1.53(n = 10) 1.85 (n = 10) HMG1 PP7435_chr2-0242 1.00 (n = 8) 1.30 (n = 8)t1HMG1^(§) PP7435_chr2-0242_trunc 1.21 (n = 8) 1.30 (n = 8) t2HMG1^(§)PP7435_chr2-0242_trunc 1.53 (n = 8) 1.30 (n = 8) ^(§)DNA-Sequences ofHMG1 were next to the full-length sequence used as two truncatedvariants (t1, t2) which were depleted of their membrane anchoringdomains that is depletion of initial 1449 nucleotides for variant t1HMG1or 1543 nucleotides in case of t2HMG1 (except their starting ATG-codon).

The yield of HyHEL-Fab produced/secreted by the engineered strainsoverexpressing a gene encoding a protein involved in involved in lipidmetabolism according to Table 4 above is increased by at least by 20% asshown in Table 4. ELO3 (PP7435_chr3-0987) was also overexpressed inCBS7435 mutS pAOX SDZ-Fab with a FC in Titer of 1.3 (n=11) and a FC inYield of 1.4 (n=11).

Example 5: Generation of Strains Underexpressing Selected Genes

P. pastoris POI production strains were engineered to comprise a knockout of a gene involved lipid biosynthesis pathway genes such as a geneinvolved in lipid storage. Selected were two genes encodingtriacylglycerol synthases: dga1 (PP7435_Chr3-1009 (Table 1) and Iro1(PP7435_Chr2-0587 Table 1)).

The P. pastoris Fab overproducing strain CBS7435 mut^(S) pAOX HyHEL-Fabwas used as host strains. A split marker cassette approach was used asdescribed by Heiss et al. (2013) [Appl Microbiol Biotechnol.97(3):1241-9.] to generate transformants with a disrupted gene locus.Verification of positive knock-out strains was done by PCR, usinggenomic DNA of transformants which had been able to grow on G418 andprimers outside of the disruption cassettes.

Table 5 lists all primers that were used for the construction of theknock-out cassettes (2 overlapping split marker cassettes per knock-outtarget): The primer pairs A_forward/A_backward, B_forward/B_backward,C_forward/C_backward, D_forward/D_backward were used to amplify thefragments A, B, C and D by PCR (Phusion Polymerase, Thermo Scientific).Fragment A is amplified from genomic P. pastoris DNA, starting 1000 bpin 5 prime direction of the respective ATG (of the targeted gene) untilapproximately 200 bp in 5 prime direction of ATG. Fragment D isamplified from genomic P. pastoris DNA, starting 200 bp in 3 primedirection of the respective ATG (of the targeted gene) until 1000 bp in3 prime direction of ATG. Fragment B consists of the first two thirds ofthe KanMX selection marker cassette and is amplified from pPM2aK21vector DNA template. Fragment B consists of the last two thirds of theKanMX selection marker cassette and is amplified from pPM2aK21 vectorDNA template. Fragments A and B are annealed together (AB) by overlapPCR using the primers A_forward and B_backward. Fragments C and D areannealed together (CD) by overlap PCR using the primers C_forward andD_backward. To generate knock-out strains, a Fab producing host strainwas transformed with total 1 μg DNA of fragments AB and CD, which bothoverlap as well. Cells were selected on YPD agar plates containing 500μg/mL G418. Positive knock-out clones were verified by PCR using theprimer pair check_forward (binds within selection marker cassette) andcheck_backward (binds in 3 prime region behind primer sequenceD_backward). Due to the replacement of a 400 bp region (around ATG) witha KanMX cassette, PCR product bands of proper size confirm integrationof the selection marker cassette at the anticipated locus.

TABLE 5 Gene identifier Primer Sequence Δdga1: A_forwardAGATATAGTTCTGTTTTATTC PP7435_Chr3- CATTAGAGGAGGATCCG SEQ 1009 ID NO: 79A_backward GTTGTCGACCTGCAGCGTACT AGATACTGGCACATAACAC SEQ ID NO: 80B_forward GTGTTATGTGCCAGTATCTAG TACGCTGCAGGTCGACAAC SEQ ID NO: 81B_backward CGGTGAGAATGGCAAAAGCTT ATG SEQ ID NO: 82 C_forwardAAGCCCGATGCGCCAGAGTTG SEQ ID NO: 83 C_backward ACCTCCTTTGCTTCTCTATCAGTGGATCTGATATCACCTA SEQ ID NO: 84 D_forward TAGGTGATATCAGATCCACTGATAGAGAAGCAAAGGAGGT SEQ ID NO: 85 D_backward ACTAACTCAGTGTCACCCAGCTC SEQ ID NO: 86 check_forward TCTTGCCATCCTATGGAACTG SEQ ID NO: 87check_backward ACAGAGCAAGACTTGCCAG SEQ ID NO: 88 ΔIro1: A_forwardACTCTAGCTGTTGTCCGCCAG PP7435_Chr2- TTC SEQ ID NO: 89 0587 A_backwardGTTGTCGACCTGCAGCGTACT ATCAATTGTGAACATAATG SEQ ID NO: 90 B_forwardCATTATGTTCACAATTGATAG TACGCTGCAGGTCGACAAC SEQ ID NO: 91 B_backwardCGGTGAGAATGGCAAAAGCTT ATG SEQ ID NO: 92 C_forward AAGCCCGATGCGCCAGAGTTGSEQ ID NO: 93 C_backward GACTCATAGAAACGACGGAAG TGGATCTGATATCACCTASEQ ID NO: 94 D_forward TAGGTGATATCAGATCCACTT CCGTCGTTTCTATGAGTCSEQ ID NO: 95 D_backward ATTCACCCAGTTAGGGCCTCC G SEQ ID NO: 96check_forward TCTTGCCATCCTATGGAACTG SEQ ID NO: 97 check_backwardTAGGAGTACCCAGCATACAG SEQ ID NO: 98

Examples 6: Combination of Genes Encoding Proteins Involved in LipidMetabolism (Over- and Underexpression)

For combinations of overexpressions of genes encoding proteins involvedin lipid metabolism, CBS7435 mut^(S) pAOX HyHEL-Fab strainsoverexpressing ELO3 (PP7435_Chr3-0987) or CBS7435 mut^(S) pAOX HyHEL-Fabstrains overexpressing PRY1 (PP7435_Chr3-1160) or CBS7435 mut^(S) pAOXHyHEL-Fab strains overexpressing HMG1 (PP7435_Chr2-0242), all of themunder control of the constitutive pGAP promoter (generated as describedin Examples 3a and b) were used as originating strains. For combinationof underexpression of genes encoding encoding proteins involved in lipidmetabolism, CBS7435 mutS pAOX HyHEL-Fab strain with a disruption in thegene locus DGA1 (Δdga1, PP7435_Chr3-1009, as described in Example 5) wasused as originating strains. In all those strains, the plasmid encodingfor the model protein (POI) HyHEL-Fab was based on Zeocin as selectionmarker, whereas the plasmids for co-overexpression of any additionalgene encoding a protein involved in lipid metabolism_or the cassettesused for disruption of the gene loci carried the KanMX resistancecassette flanked by co-directional loxP recognition sites.

Prior to transformation with an at least one further gene encoding aprotein involved in lipid metabolism_or a chaperone or a cassette usedfor disruption of the gene loci encoding a further protein involved inlipid biosynthesis, the marker gene expression cassette (KanMX— flankedby loxP sites) was recycled by Cre recombinase. Therefore, the strainsselected for marker rescue were transformed with the episomalpTAC_Cre_HphMX4 plasmid, which is expressing Cre recombinase undercontrol of S. cerevisiae TPI promoter and is transiently kept in P.pastoris as long as selection pressure by hygromycin (Hyg) is present inthe culture medium. Transformants were grown on YPD/Zeo/Hyg agar platesat 28° C. for 2 days, and replica-plated on selective agar plates forgrowth at 28° C. for further 2 days. Only clones that lost their abilityto grow on G418 and on Hyg after 2-3 plating rounds were selected for 24deep well plate (DWP) screening (described in Example 4a). Fab titer andyield were determined as described in Example 4c. The best strain interms of Fab yield and/or titer was then transformed with anotherplasmid overexpressing a further gene encoding a protein involved inlipid biosynthesis (described in Examples 3a and b) or was thentransformed with a new set of overlapping split marker knock-outcassettes (described in Example 5). 8 to 16 transformants (with twocombined proteins involved in lipid biosynthesis or knock-outs ofproteins involved in lipid biosynthesis were selected on selective agarplates (containing Zeo and G418) and screened for Fab secretion asdescribed in Example 4. For further combinatorial steps, the proceduredescribed above was repeated, thus yielding a strain with threecombinations and so on. In all screening experiments, the parental(overproducing host strain with or without empty vector) strain was usedas a reference.

a) Results on Overexpression of Combinations of Genes Encoding ProteinsInvolved in Lipid Metabolism

Combination of overexpression of at least two genes encoding proteinsinvolved in lipid metabolism in the background of overproducing strainCBS7435 mut^(S) pAOX HyHEL-Fab revealed clear improvements in terms ofFab yield and/or titer when enzymatic components of the sphingolipidbiosynthesis including fatty acid elongation pathway were overexpressedin combinations, when enzymatic components of the sphingolipidbiosynthesis including fatty acid elongation pathway were overexpressedin combination with components of the lipid transport pathway or incombination with a chaperone, when enzymatic components of the lipidtransport pathway were overexpressed in combination with components ofthe phospholipid biosynthesis pathway, or when enzymatic components ofthe ergosterol biosynthesis pathway were overexpressed in combinationwith with a chaperone.

The results are shown in Table 6

Table 6 shows the results of overexpressing combination (+) of genesencoding proteins involved in lipid metabolism_in P. pastoris. FoldChange (FC) levels for HyHEL-Fab titers/yields estimated by ELISA aregiven as relative numbers compared to the parental strain CBS7435mut^(S) pAOX HyHEL-Fab overexpressing the empty integration vector.CBS7435 mut^(S) pAOX HyHEL-Fab ELO3 was the originating strain forfurther combinations, therefore given FC values of Titer and/or FCvalues of Yield refer to this explicit clone (n=1).

TABLE 6 CBS7435 mut^(S) pAOX HyHEL-Fab Combination (+) FC of Titer FC ofYield overexpression of genes average average ELO3 1.50 (n = 1) 1.60 (n= 1) ELO3 + ELO2 1.66 (n = 8) 1.92 (n = 8) ELO3 + LAG1 1.50 (n = 10)1.60 (n = 10) ELO3 + LAG1 + LAC1 1.28 (n = 10) 1.43 (n = 10) ELO3 +LAG1 + LAC1 + 2.08 (n = 16) 2.09 (n = 16) LIP1sc* LCB1 + LCB2 1.5 (n =6) 1.1 (n = 6) ELO3 + TSC13 1.6 (n = 8) 1.8 (n=) LCB1 + LCB2 + TSC3sc*1.5 (n = 10) 1.1 (n = 10) ELO3 + LCB1 + LCB2 + 1.46 (n = 10) 1.28 (n =10) TSC3sc* ELO3 + KAR2 2.2 (n = 11 ) 2.3 (n = 11 ) ELO3 + PRY1 1.7 (n =12) 1.7 (n = 12) PRY1 + PAH1 2.2 (n = 8) 1.4 (n = 8) HMG1 + KAR2 1.4 (n= 12) 1.8 (n = 12) *sc: Saccharomyces cerevisiae

ELO3 (PP7435_chr3-0987) in combination with ELO2 (PP7435_Chr3-0603) andELO3 (PP7435_chr3-0987) in combination with PRY1 (PP7435_chr3-1160),were also overexpressed in CBS7435 mutS pAOX SDZ-Fab giving raise to aFC in Titer of 1.2 (n=12) and a FC in Yield of 1.4 (n=12), and FC inTiter of 1.3 (n=10) and a FC in Yield of 1.3 (n=10) respectively.

It can be seen that each of the combinations listed lead to an increaseof the Fab titer and/or Fab yield of the model protein HyHEL-Fab (POI)in comparison to the parental strain CBS7435 mut^(S) pAOX HyHEL-Fabtransformed with the empty vector control (see results Table 7). Theincrease of the Fab titer and/or yield in comparison to the originatingstrain indicates that combinations of genes involved in sphingolipidbiosynthesis including fatty acid elongation pathway, phospholipidbiosynthesis pathway, ergosterol biosynthesis pathway, or lipidtransport pathway, and optionally in combination with a chaperone caneven further improve the titer and/or yield of a POI exemplified by themodel protein HyHEL-Fab.

b) Results on Underexpression of Combinations of Genes Encoding ProteinsInvolved in Lipid Metabolism

For combination of two knock-outs of genes encoding proteins involved inlipid metabolism, the CBS7435 mutS pAOX HyHEL-Fab strain with adisruption in the gene loci DGA1 (PP7435_Chr3-1009) and LRO1(PP7435_Chr2-0587) was used. The strain CBS7435 mutS pAOX HyHEL-Fabwhich had the gene loci DGA1 and LRO1 successfully disrupted anddisplayed the results in terms of Fab yield and titer as shown in Table7:

Table 7 shows results of two knock-outs of genes encoding proteinsinvolved in lipid biosynthesis (lipid storage pathway). Fold Change (FC)levels for HyHEL-Fab titers and yields estimated by ELISA are given asrelative numbers compared to the parental strain CBS7435 mutS pAOXHyHEL.

TABLE 7 CBS7435 mut^(S) pAOX HyHEL-Fab Combination (+) FC of Titer FC ofYield of knock-outs average average Δdga1 + Δlro1 1.9 (n = 4) 1.44 (n =4)

Example 7: Fed Batch Cultivations

Engineered strains from Examples 3 and 6 which directly or indirectlyinfluence lipid metabolism_such as for example sphingolipid biosynthesisincluding fatty acid elongation and lipid transport were analyzed in fedbatch bioreactor cultivations for verification of production host strainimprovement.

a) Fed Batch Protocol

Respective strains were inoculated into wide-necked, baffled, covered300 mL shake flasks filled with 50 mL of YPhyG and shaken at 110 rpm at28° C. overnight (pre-culture 1). Pre-culture 2 (100 mL YPhyG in a 1000mL wide-necked, baffled, covered shake flask) was inoculated frompre-culture 1 in a way that the OD600 (optical density measured at 600nm) reached approximately 20 (measured against YPhyG media) in lateafternoon (doubling time: approximately 2 hours). Incubation ofpre-culture 2 was performed at 110 rpm at 28° C., as well.

The fed batches were carried out in 1.0 L working volume bioreactor(Minifors, Infors, Switzerland). All bioreactors (filled with 400 mLBSM-media with a pH of approximately 5.5) were individually inoculatedfrom pre-culture 2 to an OD600 of 2.0. Generally, P. pastoris was grownon glycerol to produce biomass and the culture was subsequentlysubjected to glycerol feeding followed by methanol feeding.

In the initial batch phase, the temperature was set to 28° C. Over theperiod of the last hour before initiating the production phase it wasdecreased to 25° C. and kept at this level throughout the remainingprocess, while the pH dropped to 5.0 and was kept at this level. Oxygensaturation was set to 30% throughout the whole process (cascade control:stirrer, flow, oxygen supplementation). Stirring was applied between 700and 1200 rpm and a flow range (air) of 1.0-2.0 L/min was chosen. Controlof pH at 5.0 was achieved using 25% ammonium. Foaming was controlled byaddition of antifoam agent Glanapon 2000 on demand.

During the batch phase, biomass was generated (μ˜0.30/h) up to a wetcell weight (WCVV) of approximately 110-120 g/L. The classical batchphase (biomass generation) would last about 14 hours. Glycerol was fedwith a rate defined by the equation 2.6+0.3*t (g/h), so a total of 30 gglycerol (60%) was supplemented within 8 hours. The first sampling pointwas selected to be 20 hours.

In the following 18 hours (from process time 20 to 38 hours), a mixedfeed of glycerol/methanol was applied: glycerol feed rate defined by theequation: 2.5+0.13*t (g/h), supplying 66 g glycerol (60%) and methanolfeed rate defined by the equation: 0.72+0.05*t (g/h), adding 21 g ofmethanol.

During the next 72 hours (from process time 38 to 110 hours) a total of215-216 g of methanol was supplied (with a feed rate defined by theequation 2.2+0.016*t (g/L)).

Samples were taken at various time points with the following procedure:the first 3 mL of sampled cultivation broth (with a syringe) werediscarded. 1 mL of the freshly taken sample (3-5 mL) was transferredinto a 1.5 mL centrifugation tube and spun for 5 minutes at 13,200 rpm(16,100 g). Supernatants were diligently transferred into a separatevial.

1 mL of cultivation broth was centrifuged in a tared Eppendorf vial at13,200 rpm (16,100 g) for 5 minutes and the resulting supernatant wasaccurately removed. The vial was weighed (accuracy 0.1 mg), and the tareof the empty vial was subtracted to obtain wet cell weights.

The media were as follows:

YPhyG preculture medium (per litre) contained: 20 g Phytone-Peptone, 10g Bacto-Yeast Extract, 20 g glycerol

Batch medium: Modified Basal salt medium (BSM) (per litre) contained:13.5 mL H₃PO₄ (85%), 0.5 g CaCl·2H₂O, 7.5 g MgSO₄·7H₂O, 9 g K₂SO₄, 2 gKOH, 40 g glycerol, 0.25 g NaCl, 4.35 mL PTM1, 0.1 mL Glanapon 2000(antifoam)

PTM1 Trace Elements (per litre) contains: 0.2 g Biotin, 6.0 gCuSO₄·5H₂O, 0.09 g KI, 3.00 g MnSO₄·H₂O, 0.2 g Na₂MoO₄·2H₂O, 0.02 gH₃BO₃, 0.5 g CoCl₂, 42.2 g ZnSO₄·7H₂O, 65.0 g FeSO₄·7H₂O, and 5.0 mLH₂SO₄ (95%-98%).

Feed-solution glycerol (per kg) contained: 600 g glycerol, 12 mL PTM1

Feed-solution Methanol contained: pure methanol.

b) Results

Table 8 lists the genes or gene combinations whose overexpression wasshown to increase Fab secretion/production in P. pastoris in fed batchproduction processes in comparison to the not engineered Fab producingstrains containing the empty vector (control strain). The Fab producttiter was quantified by ELISA (Example 4c). Biomass was determined aswet cell weight. Changes in product titers and yields are represented asfold change values relative to the respective control strain. Foldchange values show the improvement in titers and product yields in fedbatch production processes relative to the AOX HyHEL parental host(empty vector control) which was grown and sampled in parallel fordirect comparison.

Table 8 shows the results of overexpressing genes encoding proteinsinvolved in lipid metabolism_alone or in combination in P. pastoris infed batch cultivations. Fold Change (FC) levels for HyHEL-Fabtiters/yields estimated by ELISA are given as relative numbers comparedto the parental strain CBS7435 mut^(S) pAOX HyHEL-Fab overexpressing theempty integration vector.

TABLE 8 CBS7435 mut^(S) pAOX HyHEL-Fab Cultivation Combination (+) FC ofTiter FC of Yield Time overexpression of genes average average (h) ELO31.16 1.22 110.5 ELO3 + ELO2 1.29 1.29 112 LAG1 1.22 1.19 110 ELO3 + LAG11.89 1.63 110.5 ELO3 + LCB1 + LCB2 + 1.68 1.80 108.5 TSC3sc* PRY1 1.982.09 110.5 ELO3 + PRY1 1.69 1.84 110.5 ELO3 + KAR2 1.34 2.33 110.5 *sc:Saccharomyces cerevisiae

As shown in Table 8, all the listed genes or gene combinations succeededin increasing the yield (mg/biomass) of the model protein HyHEL-Fab byat least 20% (fold change>1.2) upon overexpression. Combinatorialoverexpression which are directly or indirectly affecting sphingolipidbiosynthesis including fatty acid elongation or metabolism or incombination with genes involved in lipid transport or a chaperone wereoutperforming the originating strain overexpressing ELO3(PP7435_chr3-0987) alone in the bioreactor.

Example 8: Lipid Analysis of Small Scale Cultivation

Biomass of P. pastoris productions strains overexpressing selected genes(listed in Table 3) produced in small scale cultivation (following thedescription in Example 4a) was used to perform lipid analysis. Afterresuspension in 10 mM Tris/HCl [pH7.5] cells were disintegrated for 10minutes with glass beads at 4° C. in a Genie Disruptor (ScientificIndustries). The resulting homogenates as well as cell debris and glassbeads were transferred to 10 ml glass Pyrex tubes and lipids wereextracted as described by Folch et al. [Folch J. et al., A simple methodfor the isolation and purification of total lipids from animal tissues,J. Biol. Chem. 226 (1957) 497-509.]. In brief, lipids were extractedwith 3 ml of CHCl₃:MeOH (2:1; v/v) vigorously shaking at roomtemperature for 1 h. Proteins and non-polar substances were removed byconsecutive washing steps with 0.2 volumes 0.034% MgCl₂, 1 ml of 2 NKCl/MeOH (4:1; v/v), and 1 ml of an artificial upper phase(CHCl₃:MeoH:H₂O, 3: 48:47; per volume). After centrifugation for 3 minat 2,000 g in a table-top centrifuge, the aqueous phase is removed byaspiration. Finally, lipids are dried under a stream of nitrogen andstored at −20° C.

For phospholipid analysis lipid extracts were loaded manually ontosilica gel 60 plates (Merck, Darmstadt, Germany). Individualphospholipids were separated by two-dimensional thin-layerchromatography using (CHCl₃/MeOH/25% NH₃, per volume) as the first, andCHCl₃/C₃H₆O/MeOH/CH₃COOH/H₂O, per volume) as the second solvent system.Phospholipids were detected by staining with iodine vapor. Stained spotswere scraped off and phospholipids were quantified by the procedure ofBroekhuyse [Broekhuyse R. M., Phospholipids in tissues of the eye Iisolation, characterization and quantitative analysis by two-dimensionalthin-layer chromatography of diacyl and vinyl-ether phospholipids,Biochim. Biophys. Acta 152 (1968) 307-315]. For total phospholipidanalysis, aliquots of dried lipid extracts were directly subjected tophosphate determination using phosphate as standard.

For analysis of non-polar lipids (sterylester (SE) and triacylglycerols(TG)) lipid extracts were loaded on Silica Gel 60 plates (Merck,Darmstadt, Germany) and separated by thin layer chromatography.Chromatograms were developed in an ascending manner by a two-stepdeveloping system. First, light petroleum/diethyl ether/acetic acid(35/15/1; per volume) was used as mobile phase, and chromatograms weredeveloped to half-distance of the plate. Then, plates were dried andchromatographs were further developed to the top of the plate usinglight petroleum/diethyl ether (49/1; v/v) as the second mobile phase.Bands were visualized by dipping the plate for 15 s into a solutionconsisting of 0.63 MnCL2.4H2O, 60 ml H2O, 60 ml methanol, and 4 mlconcentrated sulfuric acid, and incubated in a heating chamber at 105°C. for 30 min. SE and TG bands were identified and quantified bycomparison to appropriate standards (cholesteryl oleate and triolein)and densitrometric scanning at 400 nm with a Scanner (CAMAG TLC Scanner3).

Sterol analysis was performed as described by Quail and Kelly [Quail M.A., Kelly SI L., The extraction and analysis of sterols from yeast,Methods Mol. Biol. 53 (1996) 123-31]. After alkaline hydrolysis of lipidextracts using cholesterol as internal standard, gas liquidchromatography/mass spectrometry (GLC/MS) was carried out with aHewlett-Packard 5890 Gas-Chromatograph equipped with a mass selectivedetector (HP 5972), using an HP 5-MS capillary column (20 m×0.25 mmi.d.×0.25 μm film thickness). Sample aliquots of 1 μl were injected inthe splitless mode at 270° C. injection temperature with helium ascarrier gas and with a flow rate set at 0.9 ml/min in constant flowmode. The temperature program was 100° C. for 1 min, 10° C./min to 250°C., and 3° C./min to 310° C. Sterols were identified by their massfragmentation pattern.

TABLE 9 Table 9. Lipid characterization of the parental strain CBS7435mut^(S) pAOX HyHEL-Fab and the double deletion mutant Δdga1Δlro1 in therespective parental background. Lipids are listed as mg lipid(phospholipid, ergosterol, triacylglycerol, steryl ester) per g cell wetweight (CWW). If available, standard deviations are given. N.d. (notdetected). mg phospho- mg ergos- mg triacyl- mg steryl lipid/ terol/glycerol/ ester/ Strain g CWW g CWW g CWW g CWW parent 7.55 ± 0.73 0.85± 0.33 4.6 0.30 Δdga1Δlro1 5.10 ± 0.21 0.58 ± 0.21 n.d. 0.03

TABLE 10 Phospholipid pattern of total cell extracts of the parentalstrain CBS7435 mut^(S) pAOX HyHEL-Fab and the double deletion mutantΔdga1Δlro1 in the respective parental background. % of totalphospholipids Strain PI PS PC PE CL PA parent 7.52 5.41 52.11 23.09 3.838.05 Δdga1Δlro1 10.00 10.41 51.63 21.02 4.69 2.24 PI,phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine;PE, phosphatidylethanolamine; CL, cardiolipin; PA, phosphatidic acid.

Deletion of DGA1 and LRO1 results in double deletion mutant Δdga1Δlro1.DGA1 and LRO1 encode acyltransferases, which are required for thesynthesis of triacylglycerols. Both enzymes are capable to acylatediacylglycerols. DGA1 esterifies diacylglycerols in an acyl-CoAdependent manner, whereas LRO1 is independent of acyl-CoA as acyl-donor.Deletion of both triacylglycerol-acyltransferases leads to the totalloss of triacylglycerols as can be seen from Table 9. Triacylglycerolsand sterylesters are both non-polar lipids, which are involved in thestorage of excess sterols and/or fatty acids. However, their presence isnot essential for the viability of cells. Along the strong effect oftriacylglycerol depletion, the double deletion mutant Δdga1Δlro1 showsas well a strong reduction in the second class of storage lipids, thestearyl esters (Table 9). Steril esters are reduced by 10-fold comparedto the parental production strain. The depletion of triacylglycerols andthe strong reduction of stearyl esters is accompanied by a generalreduction of phospholipids and sterols (Table 9). However, individualphospholipids of the double deletion mutant Δdga1Δlro1 is not largelyaffected (Table 10). Minor changes were observed for phosphatidylserineand phosphatidylinositol that both were increased at the expense ofphosphatidic acid compared to the parental production strain.

TABLE 11 Table 11. Sterol composition of total cell extracts of theparental strain CBS7435 mut^(S) pAOX HyHEL-Fab overexpressing the emptyintegration vector (empty vector) or overexpressing genes encodingproteins involved in ergosterol biosynthesis of P. pastoris. HMG1 isoverexpressed as its native full- length version but as well as twotruncated versions (t1HMG1 and t2HMG1) both lacking their N-terminaltransmembrane domains (for details see foot note of Table 1).

indicates data missing or illegible when filed

The ergosterol biosynthetic pathway is very complex, highly regulatedand involves up to 25 enzymes. Some enzymes positioned at key roles orrate-limiting steps were chosen for overexpression. The effect on sterolcomposition and total sterol amount is displayed in Table 11.Overexpression of HMG1 as its full length variant did not lead to anychanges regarding sterol pattern and amount compared to the parentalproduction strain overexpressing the empty integration vector. However,overexpression of HMG1 as truncated versions, which are depleted fortheir N-terminal transmembrane domain, lead to an increase in theproduction of ergosterol. Highest changes in the amount of total sterolswere achieved when ERG11, a protein involved further down-stream in theergosterol biosynthetic pathway, was overexpressed.

-   -   The double deletion mutant Δdga1Δlro1 is completely depleted of        the storage lipid triacylglycerol.    -   The double deletion mutant Δdga1Δlro1 shows a strong reduction        of stearyl esters compared to the parental production strain.        Upon deletion of Δdga1Δlro1, only 10% of stearyl esters compared        to the parental production strain remain left.    -   The total amount of phospholipids is reduced to ˜65% in the        double deletion mutant Δdga1Δlro1 compared to the parental        production strain.    -   Similarly, the deletion of Δdga1Δlro1 leads to a reduction in        the total amount of sterols to roughly 68%.    -   Overexpression of ERG11 leads to the highest increase in        ergosterol and sterol precursors. The amount of ergosterol is        compared to the parental production strain overexpressing the        empty integration vector increased by 70%.

Example 9: Lipid Analysis of Fed Batch Cultivations

Cell pellets obtained at the final sampling point of bioreactorcultivations (results shown above) were used to analyse sphingolipids.Characterization of the sphingolipid distribution pattern regardingsphingolipid classes and molecular species of respective classes showeddistinct changes when helper protein ELO3 was overexpressed. The effectcaused by helper protein ELO3 overexpression could further be enhancedor altered when additional components of the sphingolipid biosyntheticprocess or lipid transport were overexpressed.

Cell pellets from fed batch cultivations were dissolved in TE-buffer (10mM Tris; 1 mM EDTA; pH 7.4) and total cell extracts prepared by vigorousshaking with glass beads at 4° C. for 15 minutes. Identical amounts (300μg protein) from total cell extracts were spiked with 30 μl of theinternal standard mix (0.15 nmol N-(dodecanoyl)-sphing-4-enine, 0.15nmol N-(dodecanoyl)-1-β-glucosyl-sphing-4-enine, 4.5 nmol C17sphinganine, Avanti Polar Lipids, Inc., Alabaster, Ala., USA), suspendedin 6 ml propan-2-ol/hexane/water (60:26:14; per vol) and incubated at60° C. for 30 min slightly modifying a protocol described previously byJ. E. Markham et al. [J. Biol. Chem. (2006). 281:22684-22694.]. Duringthe incubation, samples were shortly vortexed and sonicated after 0, 10,20 and 30 min. Then, the extracts were cleared from cell debris bycentrifugation, dried under nitrogen, redissolved in 800 μltetrahydrofuran/methanol/water (4:4:1; per vol.) [C. Buré et al., RapidCommun. Mass Spectrom. (2011). 25:3131-3145.] and stored under argon at−20° C. For analysis, samples were resolubilized by gentle heating andsonication.

UPLC-nanoESI-MS/MS was initiated by Ultra Performance LiquidChromatography (UPLC) performed on an ACQUITY UPLC® system (WatersCorp., Milford, Mass., USA) equipped with an ACQUITY UPLC® HSS T3 Column(100 mm×1 mm, 1 μm; Waters Corp., Milford, Mass., USA). Aliquots of 2 μlwere injected in the partial loop with needle overfill mode. The flowrate was 0.12 ml/min, and the separation temperature was 35° C. Inositolcontaining sphingolipids were separated by linear gradient elution asfollows: 65% solvent B held for 2 min, linear increase to 100% solvent Bfor 8 min, 100% solvent B held for 2 min and equilibration to 65%solvent B in 2 min. Ceramides (Cer) and hexosylceramides (HexCer) wereseparated as follows: 80% solvent B held for 2 min, linear increase to100% solvent B for 8 min, 100% solvent B held for 2 min andequilibration to 80% solvent B in 2 min. Solvent B wastetrahydrofuran/methanol/20 mM ammonium acetate containing 0.1% (v/v)acetic acid, and solvent A was Methanol/20 mM ammonium acetatecontaining 0.1% (v/v) acetic acid. Chip-based nanoelectrosprayionization was achieved with a TriVersa Nanomate® (Advion, Ithaca, N.Y.,USA) in the positive ion mode with 5 μm internal diameter nozzles, aflow rate of 209 nl/min and a voltage of 1.5 kV. Detection ofsphingolipid molecular species was carried out with a 4000 QTRAP® tandemmass spectrometer (AB Sciex, Framingham, Mass., USA) by monitoring (i)the transition from [M+H]⁺ molecular ions to dehydrated long chain base(LCB) fragments for Cer, HexCer and LCB; and (ii) the loss ofphosphoinositol containing head groups for inositol containingsphingolipids [C. S. Ejsing et al., J. Mass Spectrom (2006). 41:372-389.J. E. Markham et al., Rapid Commun. Mass Spectrom. (2007).21:1304-1314]. Dwell time was 30 ms and MS parameters were optimized tomaximize detector response.

Overexpression of helper protein ELO3 leads to several significantchanges in the total amount, the composition and in the molecularspecies distribution of sphingolipids. In detail, a pronounced shift inthe molecular species distribution of ceramides and the IPC-class (IPC,MIPC, M(IP)₂C) of sphingolipids was observed when helper protein ELO3was overexpressed (see Table 12 through 16). Species composition ofhexosyl-ceramides were not affected by overexpression of helper proteinELO3. Sphingolipids of the parental host strain (empty vector control)were mostly containing molecular species variants containing very longchain fatty acid (VLCFA) C24:0 in their fatty acyl moiety, whereasengineered strains overexpressing helper protein ELO3 displayed apronounced shift towards the VLCFA C26:0.

Overexpression of further components of sphingolipid biosyntheticprocesses or lipid transport did not greatly influence the molecularspecies distribution compared to the originating strain overexpressinghelper protein ELO3 (data not shown), however clear changes regardingthe relative and total abundance of certain sphingolipid classes wereobserved. Tables 17 and 18 displays the relative distribution of allsphingolipids. Overexpression of helper protein ELO3 leads to an altereddistribution pattern where the mature inositol-containingphosphorylceramide M(IP)₂C is enhanced at the expense of IPC. Allcombinations with ELO3 show a marked decrease in IPC which is eitheraccompanied by a strong increase in M(IP)₂C as in the case ofcombinations ELO3+LAG1, ELO3+KAR2, and ELO3+LCB1+LCB2+TSC3 or isaccompanied with a high increase in hexosyl-ceramides as in the case ofthe combination ELO3+PRY1.

Evaluation of helper protein ELO3 overexpression in terms of absolutevalues shows as well marked changes (see Table 18). That is,overexpression of ELO3 alone, or in combination with additional factorsinvolved in sphingolipid biosynthesis, lipid transport or chaperonesleads to a marked decrease of IPC. Simultaneously, absolute levels ofM(IP)₂C are elevated markedly when ELO3 is overexpressed. Accumulationof M(IP)₂C is even more efficiently enhanced when along to ELO3 furthercomponents are co-overexpressed. Highest accumulation of M(IP)₂C isachieved when ELO3 overexpression is combined with KAR2 overexpression.Combination of ELO3 with PRY1 leads to a general reduction in theoccurrence of complex inositol containing phosphorylceramides.

Overexpression of ELO3 leads to an altered molecular species pattern ofmost sphingolipids, that is fatty acyl moieties of ceramides andinositol-containing phosphorylceramides (IPC, MIPC, M(IP)₂C)preferentially contain C26 instead of C24 The amount of C26 is dependingon the kind of sphingolipid (ceramides, IPC, MIPC and M(IP)₂C) enhancedat least by 100% compared to the empty vector. (Tables 12-16).

TABLE 12 Composition of molecular species of ceramides from fed batchsamples of production strain CBS7436 mutS pAOX HyHEL-Fab overexpressingthe empty integration vector (empty vector) or ELO3. Results are shownas relative amounts of all ceramide species within the respectivestrains. Molecular species were excluded from the table when theirrelative amount was below 0.3% in both tested strains. Sphingolipidsconsist of a long-chain-base, which is linked via amide bond to a fattyacid. Therefore, molecular species are expressed as″(Long-chain-base/Fatty-acyl)″. Long-chain-bases and fatty acyls arefurther expressed in detail as ; Z (XX; number of carbons in the acylchain; YY: number of C—C double bonds in the acyl-chain which describesthe degree of saturation/unsaturation, Z: number of hydroxyl groups inthe chain). Table 12 Relative amount of ceramide species [%] 18:0;2/16:0; 0 18:0; 2/18:0; 0 18:0; 2/18:0; 1 18:1; 2/16:0; 0 18:1; 2/18:0;0 18:1; 2/16:0; 1 18:1; 2/18:0; 0 18:2; 2/16:0; 0 Empty vector 0.88 ±0.18 3.04 ± 0.30 0.62 ± 0.36 1.49 ± 0.36 10.24 ± 1.09 022 ± 0.11 1.88 ±0.22 0.85 ± 0.27 ELO3 0.40 ± 0.21 3.46 ± 1.21 0.99 ± 0.44 1.50 ± 0.626.17 ± 2.03 0.51 ± 0.53 2.52 ± 0.86 1.46 ± 0.52 18:2; 2/18:0; 0 18:2;2/18:0; 1 19:2; 2/18:0; 1 18:0; 3/24:0; 0 18:0; 3/26:0; 0 18:0; 3/24:0;1 18:0; 3/26:0; 1 Empty vector 4.50 ± 1.08 0.65 ± 0.28 0.81 ± 0.27 3.11± 0.30 2.55 ± 0.35 40.80 ± 0.58  27.99 ± 1.96 ELO3 4.85 ± 1.98 2.57 ±0.56 2.03 ± 0.56 0.36 ± 0.13 2.92 ± 0.53 6.19 ± 0.88 63.57 ± 7.34

TABELLE 13 Composition of molecular species of hexosyl-ceramides fromfed batch samples of production strain CBS7435 mutS pAOX HyHEL- Faboverexpressing the empty integration vector (empty vector) or ELO3 .Results are shown as relative amounts of all hexosyl-ceramide specieswithin the respective strains. Molecular species were excluded from thetable when their relative amount was below 0.3% in both tested strains.Sphingolipids consist of a long-chain-base, which is linked via amidebond to a fatty acid. Therefore, molecular species are expressed as″(Long- chain-base/Fatty-acyl)″. Long chain-bases and fatty acyls arefurther expressed in detail as XX:XY; Z (XX: number of carbons in theacyl-chain; YY: number of C—C double bonds in the acyl-chain whichdescribes the degree of saturation/unsaturation, Z: number of hydroxylgroups in the acyl- chain). Table 13 Relative amount of hexosyl-ceramidespecies [%] 18:0; 2/ 18:1; 2/ 18:2; 2/ 18:2; 2/ 19:2; 2/ 19:2; 2/ 18:0;1 18:0; 1 16:0; 1 18:0; 1 16:0; 1 18:0; 1 Empty vector 2.85 ± 3.14 ±9.67 ± 15.91 ± 6.10 ± 62.04 ± 0.20 0.21 0.62  0.81 0.34  1.10 ELO3 1.37± 2.97 ± 8.62 ± 24.14 ± 3.35 ± 59.44 ± 0.17 0.12 0.72  2.04 0.35  2.20Overexpression of ELO3 does not affect the distribution pattern ofhexosyl-ceramide species (Table 13).

TABELLE 14 Composition of molecular species ofinositolphosphorylceramides (IPC) from fed batch samples of productionstrain CBS7435 mutS pAOX HyHEL-Fab overexpressing the empty integrationvector (empty vector) or ELO3. Results are shown as relative amounts ofall IPC species within the respective strains. Molecular species wereexcluded from the table when their relative amount was below 0.3% inboth tested strains. Sphingolipids consist of a long-chain-base, whichis linked via amide bond to a fatty acid. Therefore, molecular speciesare expressed as ″(Long-chain-base/Fatty-acyl)″. Long-chain-bases andfatty acyls are further expressed in detail as XXYY; Z (XX; number ofcarbons in the acyl-chain; YY: number of C—C double bonds in the acylchain which describes the degree of saturation/unsaturation, Z: numberof hydroxyl groups in the acyl-chain). Table 14 Relative amount of IPCspecies [%] 34:0; 3 36:0; 3 36:0; 4 42:0; 3 42:0; 4 42:0; 5 44:0; 344:0; 4 44:0; 5 46:0; 2 46:0; 4 46:0; 5 Empty 0.25 ± 0.50 ± 0.43 ± 0.84± 23.01 ± 39.48 ± 0.81 ± 16.08 ± 15.64 ± 1.28 ± 1.24 ± 0.17 ± vector0.07 0.13 0.11 0.08  0.69  1.59 0.04  0.84  1.48 0.33 0.14 0.02 ELO30.39 ± 0.89 ± 0.78 ± 0.10 ±  4.87 ±  9.05 ± 1.40 ± 38.26 ± 34.49 ± 1.03± 7.09 ± 1.34 ± 0.10 0.10 0.08 0.01  0.38  0.99 0.18  1.06  0.99 0.140.47 0.21

TABELLE 15 Composition of molecular species ofmannosyl-inositolphosphorylceramides (MIPC) from fed batch samples ofproduction strain CBS7435 mutS pAOX HyHEL-Fab overexpressing the emptyintegration vector (empty vector) or ELO3. Results are shown as relativeamounts of all MIPC species within the respective strains. Molecularspecies were excluded from the table when their relative amount wasbelow 0.3% in both tested strains. Sphingolipids consist of along-chain-base, which is linked via amide bond to a fatty acid.Therefore, molecular species are expressed as ″(Long chain base/Fattyacyl)″. Long chain-bases and fatty acyls are further expressed in detailas XXYY; Z (XX: number of carbons in the acyl-chain; YY: number of C—Cdouble bonds in the acyl chain which describes the degree ofsaturation/unsaturation, Z; number of hydroxyl groups in theacyl-chain). Table 15 Relative amount of MIPC species [%] 36:0; 3 36:0;4 42:0; 3 42:0; 4 42:0; 5 44:0; 3 44:0; 4 44:0; 5 46:0; 4 Empty vector0.11 ± 0.44 ± 0.36 ± 39.31 ± 39.39 ± 0.10 ± 12.83 ±  6.28 ± 1.18 ± 0.030.15 0.11  1.72  4.20 0.04  1.74  0.73 0.22 ELO3 0.41 ± 0.77 ± 0.00 ±10.08 ±  7.62 ± 0.33 ± 59.59 ± 14.19 ± 7.02 ± 0.13 0.11 0.00  0.59  1.640.04  1.25  0.50 0.55

TABLE 16 Tabelle 16. Composition of molecular species ofmannosyl-diinositol-phosphorylceramides (M(IP)2C) from fed batch samplesof production strain CBS7435 mutS pAOX HyHEL-Fab overexpressing theempty integration vector (empty vector) or ELO3. Results are shown asrelative amounts of all M(IP)2C species within the respective strains.Molecular species were excluded from the table when their relativeamount was below 0.3% in both tested strains. Sphingolipids consist of along-chain-bases and fatty acyls are further expressed in detail asXX:YY; Z (XX: number of carbons in the acyl-chain; YY: number of C—Cdouble bonds in the acyl-chain which describes the degree ofsaturation/unsaturation, Z: number of hydroxyl groups in theacyl-chain). Relative amount of M(IP)₂C species [%]. 42:0; 4 42:0; 544:0; 4 44:0; 5 Empty 62.31 ± 4.71 16.48 ± 3.6 18.97 ± 0.77 2.24 ± 0.64vector ELO3 21.00 ± 5.74  4.43 ± 0.92 66.18 ± 7.27 8.39 ± 1.46Combining the overexpression of helper protein ELO3 with additionalhelper proteins such as LAG1, LCB1+LCB2+TSC3_(Sc), KAR2 or PRY1 leads toidentical trends in molecular species patterns of ceramides andinositol-containing phosphorylceramides (IPC, MIPC, M(IP)₂C) as thesingle overexpression of ELO3 does (less C24 containing species, moreC26 containing species). The amount of C26 is depending on the kind ofsphingolipid (ceramides, IPC, MIPC and M(IP)₂C) enhanced at least by100% compared to the empty vector. (Data not shown).

Combining the overexpression of ELO3 with additional helper proteinssuch as KAR2 enhances the shift from C24 containing species towards C26containing species in ceramides and inositol-containingphosphorylceramides (IPC, MIPC, M(IP)₂C) The amount of C26 is dependingon the kind of sphingolipid (ceramides, IPC, MIPC and M(IP)₂C) enhancedat least by 200% compared to the empty vector. (Data not shown).

The overexpression of ELO3 leads to marked changes in the relativedistribution of sphingolipids. (Table 17).

Overexpression of ELO3 leads to a reduction of IPC and MIPC ofapproximately 30% and strongly increases the formation of the matureform of inositol-containing phosphorylceramides, M(IP)₂C that iselevated by 6-fold compared to the empty vector overexpressing strain.(Table 17).

The effect of ELO3-overexpression on accumulating M(IP)₂C issynergistically enhanced by co-overexpression with either LAG1 or KAR2.M(IP)₂C is at least elevated by 10-fold compared to the empty vectoroverexpressing strain. (Table 17).

Overexpression of ELO3 does not increase the general formation ofsphingolipids. (Table 18).

Overexpression of LAG1 does not alter the fatty acyl moieties ofsphingolipids as overexpression of ELO3 does but behaves like the emptyvector control. No shift from C24 towards C26 observed. (Data notshown).

However, overexpression of LAG1 leads to similar changes in the relativedistribution pattern of sphingolipids, that is, increase in M(IP)₂C atthe expense of IPC. (Table 17).

Overexpression of LAG1 increases the formation of M(IP)2C by at least10-fold compared to the empty vector overexpressing strain. (Table 18).

Overexpression of PRY1 does neither show changes in the speciesdistribution nor in the sphingolipid distribution. (Table 17).

Overexpression of PRY1 shows in general reduced levels ofinositol-containing phosphorylceramides (less IPC, MIPC and M(IP)₂C).(Table 18).

TABELLE 17 Sphingolipid analysis from fed batch samples of productionstrain CBS7435 mutS pAOX HyHEL-Fab overexpressing the empty integrationvector (empty vector) or overexpressing, ELO3, ELO3 LAG1, LAG1, ELO3LCB1 LCB2 TSC3Sc, PRY1, ELO3 PRY1 or ELO3 KAR2. Results show therelative distrubution of all sphingolipid classes (Cer, ceramide;HexCer, hexosyl-ceramide; IPC, inositolphosphorylceramide; MIPC,mannosyl-inosilolphophorylceramide; M(IP)2C,mannosyl-diinositol-phosphorylceramide) within the respective stains.Table 17 %-total sphingolipids ELO3 LC81 Empty vector ELO3 ELO3 LAG1LAGI LCB2 TSC3_(Sc) PRY1 ELO3 PRY1 ELO3 KAR2 Cer 0.74 3.55 3.62 2.983.55 1.68 1.09 4.67 HexCer 30.06 35.00 37.18 24.58 41.10 39.65 50.6025.19 IPC 54.72 38.06 25.15 42.77 35.31 40.29 35.13 32.34 MIPC 12.158.51 9.15 7.20 5.11 13.52 7.28 6.02 M(IP)₂C 2.33 14.89 24.91 22.47 14.934.86 2.89 31.78

TABELLE 18 Comparison of absolute values of analyzed sphingolipids fromfed batch samples of production strain CBS7435 mutS pAOX HyHEL- Faboverexpressing the empty integration vector (empty vector) oroverexpressing, ELO3, ELO3 LAG1, LAG1, ELO3 LCB1 LCB2 TSC3Sc, PRY1, ELO3PRY1 or ELO3 KAR2. Identical amounts of sample were analyzed.Presentation of peak intensities enables the direct comparison of therelative abundance all sphingolipid classes (Cer, ceramide: HexCer,hexosyl-ceramide: IPC, inositolphosphorylceramide; MIPC, mannosyl-inositolphosphorylceramide M(IP)2C,mannosyl-diinositol-phosphorylceramide) amongst the analyzed strains.Table 18 Peak area (counts) ELO3 LCB1 Empty vector ELO3 ELO3 LAG1 LAG1LCB2 TSC3_(Sc) PRY1 ELO3 PRY1 ELO3 KAR2 Cer 38546 160685 156753 223371124251 64033 118798 287553 HexCer 1555970 1585610 1612210 18444101437820 1506930 1469250 1551850 IPC 2832338 1724000 1090460 37086871235200 1531320 1020090 1991970 MIPC 628725 385407 396573 540399 178834513662 211342 370634 M(IP)₂C 120482 674311 1080140 1685574 522132 18464983904 1957911

1. A method of increasing the yield of a protein of interest in aeukaryotic host cell, comprising overexpressing in said host cell atleast one polynucleotide encoding at least one protein which is involvedin lipid metabolism, thereby increasing the yield of said protein ofinterest in comparison to a host cell which does not overexpress apolynucleotide encoding a protein which is involved in lipid metabolism,wherein said protein which is involved in lipid metabolism is involvedin sphingolipid biosynthesis and comprises an amino acid sequence asshown in SEQ ID NO: 1 or a homologue thereof having at least 70%sequence identity to an amino acid sequence as shown in SEQ ID NO: 1,wherein 1, 2, 3, 4, 5, 6, 7, 8, or more of the proteins involved inlipid metabolism selected from SEQ ID NO: 1 or homologues thereof,wherein the homologues have at least 70% sequence identity to an aminoacid sequence as shown in SEQ ID NO: 1, are overexpressed, and whereinat least one protein involved in lipid transport is also overexpressed,or wherein at least one polynucleotide encoding chaperone is alsooverexpressed.
 2. A method of manufacturing a protein of interestaccording to claim 1, comprising providing the host cell engineered tooverexpress at least one polynucleotide encoding at least one protein,wherein said protein which is involved in lipid metabolism is involvedin sphingolipid biosynthesis and comprises an amino acid sequence asshown in SEQ ID NO: 1 or a homologue thereof, wherein the homologue hasat least 70% sequence identity to an amino acid sequence as shown in SEQID NO: 1, wherein said host cell comprises a heterologous polynucleotideencoding a protein of interest; culturing the host cell under suitableconditions to overexpress the protein involved in lipid metabolism orhomologue thereof and to express said protein of interest; andoptionally isolating said protein of interest from the cell culture,wherein 1, 2, 3, 4, 5, 6, 7, 8 or more of the proteins involved in lipidmetabolism selected from SEQ ID NO: 1 or homologues thereof, wherein thehomologues have at least 70% sequence identity to an amino acid sequenceas shown in SEQ ID NO: 1, are overexpressed, and wherein at least oneprotein involved in lipid transport is also overexpressed, or wherein atleast one polynucleotide encoding chaperone is also overexpressed. 3.The method of claim 1, wherein said eukaryotic host cell is a fungalhost cell.
 4. The method of claim 3, wherein said eukaryotic host cellis selected from the group consisting of Pichia pastoris, Hansenulapolymorpha, Trichoderma reesei, Saccharomyces cerevisiae, Kluyveromyceslactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii,Komagataella sp., Aspergillus sp. and Schizosaccharomyces pombe.
 5. Themethod of claim 1, wherein said protein involved in lipid transportcomprises an amino acid sequence as shown in SEQ ID NO: 11 or ahomologue thereof, wherein the homologue has at least 70% sequenceidentity to an amino acid sequence as shown in SEQ ID NO: 11,respectively.
 6. The method of claim 1, wherein said chaperone comprisesan amino acid sequence as shown in SEQ ID NO: 18 or a homologue thereof,wherein the homologue thereof comprises at least 70% sequence identityto an amino acid sequence as shown in SEQ ID NO: 18, respectively. 7.The method of claim 1, wherein said protein of interest is anon-membrane protein of interest.
 8. The method of claim 7, wherein saidnon-membrane protein of interest is any one of an enzyme, a therapeuticprotein, a food additive or feed additive.
 9. The method of claim 8,wherein said therapeutic protein comprises an antibody or an antibodyfragment still having the activity of binding its antigen.
 10. Themethod of claim 1, wherein said protein involved in sphingolipidbiosynthesis or homologue thereof increases the yield of the modelprotein HyHEL (SEQ ID NO: 39 for heavy chain and SEQ ID NO: 40 for lightchain) compared to the host cell prior to engineering by at least 10%,at least 15%, at least 20%, at least 25%, at least 30%.
 11. The methodof claim 2, wherein said eukaryotic host cell is a fungal host cell. 12.The method of claim 11, wherein said eukaryotic host cell is selectedfrom the group consisting of Pichia pastoris, Hansenula polymorpha,Trichoderma reesei, Saccharomyces cerevisiae, Kluyveromyces lactis,Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataellasp., Aspergillus sp. and Schizosaccharomyces pombe.
 13. The method ofclaim 2, wherein said protein involved in lipid transport comprises anamino acid sequence as shown in SEQ ID NO: 11 or a homologue thereof,wherein the homologue has at least 70% sequence identity to an aminoacid sequence as shown in SEQ ID NO: 11, respectively.
 14. The method ofclaim 2, wherein said chaperone comprises an amino acid sequence asshown in SEQ ID NO: 18 or a homologue thereof, wherein the homologuethereof comprises at least 70% sequence identity to an amino acidsequence as shown in SEQ ID NO: 18, respectively.
 15. The method ofclaim 2, wherein said protein of interest is a non-membrane protein ofinterest.
 16. The method of claim 15, wherein said non-membrane proteinof interest is any one of an enzyme, a therapeutic protein, a foodadditive or feed additive.
 17. The method of claim 16, wherein saidtherapeutic protein comprises an antibody or an antibody fragment stillhaving the activity of binding its antigen.
 18. The method of claim 2,wherein said protein involved in sphingolipid biosynthesis or homologuethereof increases the yield of the model protein HyHEL (SEQ ID NO: 39for heavy chain and SEQ ID NO: 40 for light chain) compared to the hostcell prior to engineering by at least 10%, at least 15%, at least 20%,at least 25%, or at least 30%.